Can you read the article and follow the instructions in the Powerpoint. You can write the answers directly in the powerpoint or in a separate paper. As the questions in the powerpoint said what is the citations ? what the material and methods which means what the cell lines, what were the reagents and drugs if were any used and what were the major assays?

 

Finally, Complete each panel in each Figure with

1) cell lines or models- if not shown in the Figures

2) Key points with arrows or boxes in each figure there is a, b, c etc so in each one of those make an arrow or box for the important part in it and explain why from the paper. 

3) what assay used

4) what drug or other treatments if not shown in the fig 

5) if there was an abbreviations used spelled out 

Huang et al. Cell Death Discovery (2020) 6:70
https://doi.org/10.1038/s41420-020-00301-2 Cell Death Discovery

A R T I C L E Op e n A c c e s s

BECN1 promotes radiation-induced G2/M arrest
through regulation CDK1 activity: a potential role
for autophagy in G2/M checkpoint
Ruixue Huang1, Shanshan Gao2, Yanqin Han2, Huacheng Ning1,2, Yao Zhou1,2, Hua Guan2, Xiaodan Liu2,
Shuang Yan2 and Ping-Kun Zhou2,3

Abstract
Authophagy and G2/M arrest are two important mechanistic responses of cells to ionizing radiation (IR), in particular
the IR-induced fibrosis. However, what interplayer and how it links the autophagy and the G2/M arrest remains elusive.
Here, we demonstrate that the autophagy-related protein BECN1 plays a critical role in ionizing radiation-induced G2/
M arrest. The treatment of cells with autophagy inhibitor 3-methyladenine (3-MA) at 0–12 h but not 12 h
postirradiation significantly sensitized them to IR, indicating a radio-protective role of autophagy in the early response
of cells to radiation. 3-MA and BECN1 disruption inactivated the G2/M checkpoint following IR by abrogating the IR-
induced phosphorylation of phosphatase CDC25C and its target CDK1, a key mediator of the G2/M transition in
coordination with CCNB1. Irradiation increased the nuclear translocation of BECN1, and this process was inhibited by
3-MA. We confirmed that BECN1 interacts with CDC25C and CHK2, and which is mediated the amino acids 89–155 and
151–224 of BECN1, respectively. Importantly, BECN1 deficiency disrupted the interaction of CHK2 with CDC25C and the
dissociation of CDC25C from CDK1 in response to irradiation, resulting in the dephosphorylation of CDK1 and
overexpression of CDK1. In summary, IR induces the translocation of BECN1 to the nucleus, where it mediates the
interaction between CDC25C and CHK2, resulting in the phosphorylation of CDC25C and its dissociation from CDK1.
Consequently, the mitosis-promoting complex CDK1/CCNB1 is inactivated, resulting in the arrest of cells at the G2/M
transition. Our findings demonstrated that BECN1 plays a role in promotion of radiation-induced G2/M arrest through
regulation of CDK1 activity. Whether such functions of BECN1 in G2/M arrest is dependent or independent on its
autophagy-related roles is necessary to further identify.

Introduction
Radiotherapy is a widely used strategy for the treatment of

cancer patients. However, despite major advances in radio-
therapy, the radioresistance of tumors remains the leading
obstacle to their clinical treatment because it results in
radiotherapy failure or tumor recurrence1. Approximately

10–45% of cancers are resistant to radiation, which greatly
influences the outcomes of radiotherapy.
Autophagy is a process of cellular self-degradation that

plays a critical role in maintaining the balance between
cell survival and cell death2. Recent studies have indicated
that the two roles of autophagy in cancer cells are asso-
ciated with the initiation of a cascade of signaling path-
ways involving multiple molecules and transcription
factors3,4. The suppression of BECN1 phosphorylation
reduces autophagy in prostate cancer cells and thereby
overcomes the radioresistance of these cells5. Conversely,
the autophagy-mediated degradation of p62 and the
c-Jun-mediated expression of BECN1 increase the

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Correspondence: Ping-Kun Zhou (birm4th@163.com)
1Department of Occupational and Environmental Health, Xiangya School of
Public Health, Central South University, 410078 Changsha, Hunan Province,
China
2Department of Radiation Biology, Beijing Key Laboratory for Radiobiology,
Beijing Institute of Radiation Medicine, AMMS, 100850 Beijing, China
Full list of author information is available at the end of the article
Edited by Ivano Amelio

Official journal of the Cell Death Differentiation Association

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mailto:birm4th@163.com

radioresistance of lung cancer cells6. We established
BECN1-knockout human triple-negative breast cancer
(TNBC) MDA-MB-231 cells using the CRISPR/
Cas9 system, and functional analyses revealed that

BECN1

deficiency suppressed MDA-MB-231 cell proliferation by
inducing arrest of the cell cycle at the G1 phase in vitro

7,
which suggested that BECN1 regulates the cell cycle.
The activation of cell-cycle checkpoints is another

fundamental process for controlling cellular homeostasis
during exposure to radiation. During the cell cycle, var-
ious checkpoints play an important role in monitoring
and regulating the progression as well as the genomic
stability in response to DNA damage or other stresses. Li
et al. reported that radiation induces G2/M phase arrest in
primary human renal clear cell carcinoma cells, which
might contribute to radioresistance8. Carruthers et al.
found that the suppression of ATM kinase, an upstream
kinase of the G2/M checkpoint signal pathway involving
CHK2/CDC25C/CDK1, by the inhibitor KU-55933 inac-
tivates the G2/M checkpoint and radiosensitizes glio-
blastoma stem-like cells9.
In the present study, we focused on BECN1 based on

the following considerations. BECN1, the mammalian
homolog of yeast Atg6 and a highly conserved eukaryotic
protein10, is a core autophagy-related protein that reg-
ulates vesicle nucleation during autophagosome forma-
tion. The blockage of autophagy using the autophagy
inhibitor 3-methyladenine (3-MA) increases the number
of cells in the G2/M state

11. BECN1 also associates with
class III phosphoinositide 3-kinase (PI3K) to promote the
generation of phosphatidylinositol 3-phosphate12. BECN1
is also involved in the cellular response to radiation-
induced damage or sensitivity13. Moreover, our previous
study showed that the knockout of BECN1 suppresses
TNBC cell proliferation and colony formation by inducing
cell-cycle arrest at the G1 phase

7. Our pilot study revealed
that the inhibition of autophagy by 3-MA decreased the
number of LC3 puncta and LC3B protein expression in γ-
ray-irradiated cells (Fig. 1a). Thus, we hypothesized that
autophagy-related BECN1 might play a markedly more
important role in the activation of cell-cycle checkpoints
in response to irradiation. Our findings demonstrated that
BECN1 plays a role in promotion of radiation-induced
G2/M arrest through regulation of CDK1 activity. Whe-
ther such functions of BECN1 in G2/M arrest is depen-
dent or independent on its autophagy-related roles is
necessary to further identify.

Results
BECN1 protein was remarkably upregulated in A549 cells,
Hela cells, and mice lung fibrosis tissues
We performed immunofluorescence detection of the

autophagy-related markers LC3 and WIPI2 (WD-repeat
domain, phosphoinositide interacting 2), which is a

member of the ATG18/WIPIs (WD-repeat protein
interacting with phosphoinositide) family14. As shown in
Fig. 1a, b, c, upregulation of BECN1 was unexpectedly
observed in these cell lines, revealing the potential asso-
ciation of autophagy with radiation-related development
of lung fibrosis. After 4 Gy irradiation, the protein levels of
LC3-II were significantly increased overtime after IR,
whereas the SQSTM1/P62 level was decreased; this trend
was largely reversed to a certain extent by 3-MA treat-
ment (Fig. 1d). The numbers of LC3 puncta per cells
increased postradiation, but this effect was inhibited by
treatment with 3-MA and wortamannin (Fig. 1e). As
shown in Fig. 1f, g, the numbers of LC3 and WIPI2
puncta/dots increased postradiation, but this effect was
inhibited by treatment with 3-MA. The level of BECN1 in
mice radiation-induced lung fibrosis tissues are remark-
ably upregulated compared with the control group with-
out radiation treatment (Fig. 2a). IHC staining was
conducted and the BECN1 expression was verified to be
upregulated in ice radiation-induced lung fibrosis tissues
compared with the control group without radiation
treatment (Fig. 2b). Collectively, these above data strongly
demonstrate that autophagy and BECN1 protein expres-
sion are elevated in human cells in vitro, and mouse lung
fibrosis tissues in vivo.

BECN1 and its mediated autophagy were essential for cell
response to radiation and lung fibrosis
At the early stage (0–12 h) after irradiation, a series of

survival-promoting DDRs, such as cell-cycle checkpoints
and DNA repair, are activated and encourage cells to
recover from radiation damage. Therefore, we designed
experiments in which the cells were treated with the
autophagy inhibitor 3-MA at the early (0–12 h post-IR) or
later stages (12–24 h post-IR). Supplementary Fig. 1a
suggest that autophagy might play a protective role during
the early stage (0–12 h) but not at the later stage of the
response to IR. After 2- or 4-Gy irradiation, the survival
rates of 3-MA-treated HeLa cells were significantly
decreased compared with those of the untreated cells
(Supplementary Fig. 1b). As shown in Supplementary Fig.
1c, d, the apoptosis level was markedly increased in the
group treated with 3-MA at 0 to 12 h post 4-Gy-
irradiation (4 Gy + 3-MA@0 h) compared with the
group that was only irradiated with 4 Gy (4 Gy). These
results further suggest that autophagy plays a dual-effect
role in the early response of cells to irradiation, which at
the early stage (0–12 h) of radiation exposure, autophagy
is subjected to protective effect but at the later stage
(12–48 h), it is subjected to inhibition effect.
The LC3-II level increased with increasing time post-IR,

but a notably lower increase was observed in the cells
cotreated with 3-MA (Fig. 2c). Autophagic flux was found
to be active in irradiated BECN1-WT cells. BECN1

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deficiency resulted in a significantly decreased LC3B II/I
ratio in BEC1-KO cells compared with BECN1-WT cells
(Fig. 2d). We used cells transfected with the tandem GFP-
LC3-mRFP-LC3ΔG plasmid to further confirm the effect
of BECN1-KO on autophagic flux. This fusion protein can
be cleaved by the endogenous ATG4 family protease to
generate equal amounts of GFP-LC3 and mRFP-LC315,16.
In the acidic environment of the lysosome, mRFP-LC3

(red) is markedly more stable than GFP-LC3 (green).
MRFP red puncta represent the maturation of autopha-
golysosomes, and yellow puncta (indicating the expres-
sion of both GFP and mRFP) represent the formation of
autophagosomes. A lower ratio of GFP-LC3 to RFP-LC3
puncta reflects autophagic flux. Both yellow and red
puncta were observed in the cells, and a decreased ratio of
GFP-LC3 to RFP-LC3 puncta was apparent following

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F G

Fig. 1 BECN1 was overexpressed in MDA-MB-231, A549, and mouse tissues post-exposure of IR. a qRT-PCR was performed to detect BECN1
mRNA expression and the asterisk (*) indicates a significant increase post 4 Gy IR exposure compared to the non-IR exposure in MDA-MB-231 cells.
b qRT-PCR was performed to detect BECN1 mRNA expression and the asterisk (*) indicates a significant increase post 4 Gy IR exposure compared to
the non-IR exposure in A549 cells. c Western blot assay was performed to detect theATG3 and BECN1 expression in MDA-MB-231 cells (n = 5) post
4 Gy IR exposure. d The protein levels of LC3 and SQSTM1/P62 in MDA-MB-231 cells were detected by Western blotting analysis. GAPDH was used as
the loading control. MDA-MB-231 cells cotreated or not cotreated with 3-MA were harvested at the indicated timepoints after 4-Gy γ-ray-irradiation.
e MDA-MB-231 cells cotreated or not cotreated with 3-MA or wortamannin were harvested at the indicated timepoints after 4-Gy γ-ray-irradiation.
The number of LC3 puncta per cell. was analyzed and the asterisk (*) indicates a significant change compared with control group. f Autophagic GFP-
LC3 puncta in 4-Gy γ-ray-irradiated MDA-MB-231 cells cotreated with or without 3-MA were detected by fluorescence confocal microscopy(bars: ×20
magnification). g Autophagic WIPI2in 4-Gy γ-ray-irradiated MDA-MB-231 cells cotreated with or without 3-MA was detected by fluorescence confocal
microscopy(bars: ×20 magnification). The data are presented as the means ± SDs from three independent experiments *p < 0.01 compared with the control group.

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4-Gy irradiation (Fig. 2e). However, fewer LC3 puncta and
a higher ratio of GFP-LC3 to RFP-LC3 puncta were
observed in the irradiated BECN1-KO cells, particularly at
12 h, compared with the BECN1-WT cells (Fig. 2e, f).

As shown in Fig. 2g, at indicated timepoints after 4 Gy IR
treatment, the E-cadherin expression level was increased
and E-cadherin and Vimentin expression levels were
decreased at the BECN1 deficiency status. These data

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Fig. 2 (See legend on next page.)

Huang et al. Cell Death Discovery (2020) 6:70 Page 4 of 17

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reveal that BECN1-mediated autophagy is at least one of
key events for BECN1 promotion of radiation-induced
EMT.

G2/M checkpoint was regulated by BECN1 in response to IR
As shown in Fig. 3a, b, the percentages of G2- and M-

phase cells increased in a time-dependent manner after 6-
Gy irradiation, which indicated the induction of G2/M
arrest and/or mitotic arrest. The cells pretreated with 3-
MA exhibited relatively lower percentages of cells at the
G2 and M phases compared with those found for the cells
that were only irradiated alone (Fig. 3b). A similar result
was also observed after pretreatment with chloroquine,
another autophagy inhibitor (Supplementary Fig. 2).
Because autophagy might participate in IR-induced cell-
cycle arrest, we hypothesized that BECN1, a key regulator
of autophagy, might play a critical role in the crosstalk
between autophagy and cell-cycle progression. CRISPR/
Cas9-mediated BECN1-knockout MDA-MB-231 cells
(BECN1-KO) were used (Fig. 3d). As shown in Fig. 3c, e,
BECN1 deficiency resulted in decreased G2/M arrest in
response to IR compared with wild-type (WT) BECN1.
Rescue experiments further showed that the restoration of
BECN1 might increase G2/M arrest in response to IR. The
silencing of BECN1 expression by specific siRNAs (Sup-
plementary Fig. 3a) further demonstrated that autophagy-
related BECN1 is involved in the regulation of irradiation-
induced G2/M arrest (Supplementary Fig. 3b, c).
CRISPR/Cas9-mediated ATG7-knockout MDA-MB-

231 cells were also used to further investigate the cross-
talk between autophagy and cell-cycle progression, and
the knockout of ATG7 expression was detected by wes-
tern blotting (Fig. 3g). As shown in Fig. 3f, h, ATG7
deficiency resulted in decreased G2/M arrest in response
to IR, which was similar to the effect observed with
BECN1 deficiency.
To further clarify the role of autophagy-related BECN1

in radiation-induced G2/M arrest, the cells were analyzed
by flow cytometry following the immunostaining of

phospho-histone H3/Ser10 (pHH3), a marker of cells in
mitosis17,18. As shown in Fig. 3a, the ratio of pHH3-
positive mitotic cells was sharply decreased in the popu-
lation of control cells at 2–4 h postirradiation, which
indicated that activation of the G2/M boundary check-
point results in arrest at the G2/M transition. Eight hours
post-IR, the mitotic cells in the population of cells treated
with radiation and 3-MA was scarcely decreased (Fig. 4a,
b). PHH3 immunofluorescence staining was also per-
formed in BECN1-WT and BECN1-KO MDA-MB-231
cells (Fig. 4c, d). The proportion of pHH3-positive mitotic
cells was sharply decreased in the population of BECN1-
WT cells at 2–4 h postirradiation. In contrast, the pro-
portion of mitotic cells in the population of

BECN1-KO

MDA-MB-231 cells was unaffected at the early stage
(0–8 h) of the response to IR. At 12 h postirradiation, an
increased proportion of mitotic BECN1-KO cells were
observed, which suggested the occurrence of prolonged
mitotic arrest in the population of BECN1-deficient cells.
A representative image of MPM2-positive mitotic cells is
shown in Fig. 4e. The MPM2 staining results further
indicated that BECN1 deficiency liberated cells after IR-
induced G2/M arrest, i.e., BECN1 deficiency resulted in
inactivation of the G2/M checkpoint (Fig. 4e, f). Thus, the
results clearly demonstrate that autophagy-associated
BECN1 deficiency inactivates the G2/M checkpoint in
response to IR.

BECN1 deficiency disrupts the responses of G2/M
checkpoint proteins to IR
As shown in Fig. 5a, b, irradiated HeLa cells cotreated

with 3-MA exhibited decreased levels of pATM, pCHK2,
pCDC25C, and pCDK1 at the indicated timepoints post-
irradiation compared with the control cells.

CDK1

dephosphorylation and CCNB1 expression are required
for activation of the CDK1-CCNB1 complex19. This
complex is maintained in an inactive state through
phosphorylation of a conserved residue, tyrosine 15
(CDK1-pY15)20. As shown in Supplementary Fig. 4a, b,

(see figure on previous page)
Fig. 2 Involvement of autophagy in the regulation of IR-induced EMT. a The BECN1 expression level was analyzed in 20 paired mice lung fibrosis
tissues with 20 Gy radiation and their normal tissues without 20 Gy radiation by calculating the integrated IOD/area using Image-Pro Plus version 6.0.
Three independent experiments were performed, the student’s t-test was utilized to determine the p-value and the asterisk (*) indicates a significant
change compared with control group. b IHC staining was performed to evaluate BECN1 expression in 20 paired mice lung tissues with 20 Gy
radiation and their normal tissues without 20 Gy radiation. The IHC images were captured using the AxioVision Rel.4.6 computerized image system.
c An autophagy flux experiment was performed to test the autophagy process. The protein levels of BECN1 and LC3B in MDA-MB-231 cells were
detected by western blotting analysis. GAPDH was used as the control. MDA-MB-231 cells cotreated with or without 3-MA were harvested at the
indicated timepoints after 4-Gy γ-ray-irradiation. d An autophagy flux experiment was performed to test the autophagy process. The protein levels of
BECN1 and LC3B in MDA-MB-231 cells were detected by western blotting analysis. GAPDH was used as the control. MDA-MB-231 cells cotreated with
or without 3-MA were harvested at the indicated timepoints after4-Gy γ-ray-irradiation. e The effect of BECN1 knockout on autophagy flux was
examined. The cells were transfected with tandem GFP-LC3-mRFP-LC3ΔG plasmids mediated by the adenovirus, and fluorescence laser confocal
microscopy was performed(bars: ×20 magnification). f Quantitative measurement of GFP and mRFP. The data are presented as the means ± SDs from
three independent experiments; *p < 0.05 compared with the WT cells. g Western blot assay was performed to detect the radiation-induced EMT biomarkers’ alteration post 4 Gy radiation in MDA-MB-231 cells at indicated timepoints.

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Fig. 3 Involvement of autophagy in the regulation of IR-induced cell-cycle progression arrest. a Representative flow cytometry histogram of
cell-cycle progression in the population of 4-Gy γ-ray-irradiated HeLa cells with or without cotreatment with 3-MA. b Effect of 3-MA on the G2 and M
phase distribution in the population of irradiated cells. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 compared with the control group. c Representative flow cytometry histogram of cell-cycle progression in the population of 4-Gy γ-ray-irradiated BECN1-WT and BECN1-KO MDA-MB-231 cells. d Western blotting analysis of BECN1 in BECN1-WT MDA-MB-231 cells (control) and BECN1-KO cells generated by CRISPR/Cas9. GAPDH served as the internal loading control. e Quantitative measurement of the proportions of 4-Gy γ-ray-irradiated BECN1-KO and control cells in the G2 and M phases at the indicated times after IR. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 compared with the control group. f Representative flow cytometry histogram of cell-cycle progression in the population of 4-Gy γ-ray-irradiated ATG7-WT and AG7-KO MDA-MB-231 cells. g Western blotting analysis of ATG7 in ATG7-WT MDA-MB-231 cells (control) and ATG7-KO cells generated by CRISPR/Cas9. GAPDH served as the internal loading control. h Quantitative measurement of the proportion of 4-Gy γ-ray-irradiated ATG7-KO and control cells in the G2 and M phases at the indicated times after IR. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 compared with the control group.

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Time post-irradiation 0 2h 4h 6h 8h 12h

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MPM2 DAPI Merge MPM2 DAPI Merge

Mitotic cell 1 Mitotic cell 2

20 m20 m 20 m 20 m 20 m 20 m

Fig. 4 Autophagy-related BECN1 deficiency inactivates the G2/M checkpoint in response to IR. a Representative flow cytometry histograms of
phosphorylated histone H3 (pHH3)-positive mitotic cells in the population of γ-ray-irradiated HeLa cells with or without 3-MA cotreatment. The cells
were immunostained with pHH3(Ser10) antibody to detect the proportion of pHH3-positive mitotic cells at the indicated times after IR. b
Quantitative measurement of pHH3(Ser10)-positive cells. The data are presented as the means ± SDs from three independent experiments; *p < 0.01 compared with the control group. c Representative flow cytometry histogram of the population of γ-ray-irradiated BECN1-WTor BECN1-KO MDA-MB- 231 cells. The cells were immunostained with pHH3(Ser10) antibody to detect the proportion of pHH3-positive cells at the indicated times after IR. A rescue experiment was conducted by transfecting BECN1-KO MDA-MB-231 cells with lentiviral vectors expressing BECN1. d Quantitative measurement of pHH3(Ser10)-positive cells. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 compared with the WT cells. e Representative image of mitotic protein monoclonal-2 (MPM2)-positive cells obtained through immunofluorescence staining (bars: ×20 magnification). f Quantification of MPM2 antibody-stained mitotic cells. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 compared with the WT cells. g pHH3 was detected by Western blotting analysis. β-actin served as the internal control.

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Fig. 5 Autophagy inhibition and BECN1 deficiency disturb the response of G2/M checkpoint-related proteins to IR. a Western blotting
analysis of G2/M checkpoint-regulating proteins (pATM, pCHK2, and CHK2) in the γ-ray-irradiated A549 cells that were cotreated or not cotreated with
the autophagy inhibitor 3-MA. The proteins were detected at the indicated timepoints after IR, and GAPDH served as the internal control. b Western
blotting analysis of the phosphorylation of G2/M checkpoint-regulating proteins (pCDC25C, pCDK1, and CDK1) in the γ-ray-irradiated A549 cells with
or without cotreatment with the autophagy inhibitor 3-MA. The phosphorylation analysis was performed at the indicated timepoints after IR, and
GAPDH served as the internal control. c Western blotting analysis of G2/M checkpoint-associated phosphorylated proteins (pCHK1, CHK1, pWEE1, and
pMYT1) in the γ-ray-irradiated BECN1-WTor BECN1-KO MDA-MB-231 cells. GAPDH served as the internal control. d Western blotting analysis of G2/M
checkpoint-associated phosphorylated proteins (pCHK2, CHK2, pCDK1, and CDK1) in the γ-ray-irradiated BECN1-WTor BECN1-KO MDA-MB-231 cells.
GAPDH served as the internal control. e Quantitative measurement of the levels of pATM, pCHK2, pCDC25C, and pCDK1 in A549 cells with or without
3-MA cotreatment detected at the indicated time point after IR. The data are presented as the means ± SDs from three independent experiments; *p
< 0.05 between different groups. f Quantitative measurement of phosphorylated protein levels of pCHK1, pWEE1, pMYT1, and pCDK1 in

BECN1-WT

and BECN1-KO MDA-MB-231 cells at the indicated timepoints after IR. The data are presented as the means ± SDs from three independent
experiments; *p < 0.05 between different groups.

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the proportion of pHH3-positive mitotic cells was sharply
decreased in the population of WT cells at 2–4 h post-
irradiation, whereas the numbers of mitotic cells in the
populationofATG7-KO MDA-MB-231 cells was unaf-
fected. ATG7 deficiency resulted in decreased G2/M
arrest in response to IR. A rescue experiment showed that
restoration of the expression of ATG7 could liberate cells
from G2/M arrest in response to IR. Compared with
WT cells, a notably lower increase in LC3-II and slower
degradation of p62 protein were observedATG7 KO cells
with increasing irradiation time (Supplementary Fig. 4c).
The levels of phosphorylated pCHK1, pCHK2, pWEE1,

and pCDK1 were increased in BECN1-WT MDA-MB-
231 cells after 6-Gy irradiation, whereas no obvious
change or only a weak increase in the levels of nonpho-
sphorylated CHK2, WEE1, and CDK1 were detected in
BECN1-WT MDA-MB-231 cells. Overall, the expression
of MYT1 in BECN1-KO cells was lower than that in
WT cells at all examined times postirradiation (Supple-
mentary Fig. 5a, b). The changes in the phosphorylation
levels of the related proteins are shown in Fig. 5c, d.
Lower levels of pCHK1, pCHK2, pWEE1, pMYT1, and
pCDK1 were observed in BECN1-KO MDA-MB-231 cells
compared with BECN1-WT cells at the indicated time-
points postirradiation. These results suggest that (i) CDK1
is phosphorylated and inactivated in WT cells following
irradiation but is maintained in a dephosphorylated state
in BECN1-KO cells; (ii) BECN1 deficiency might promote
the dephosphorylation of CDK1; and (iii) Inactivation of
the G2/M checkpoint due to autophagy inhibition or
BECN1 deficiency might be partially due to dysregulation
of the ATM/CHK2/CDC25C/CDK1 signaling pathway.

BECN1 deficiency disrupts the dissociation of CDC25C from
CDK1 following irradiation
As shown in Fig. 6a, the phosphorylated CDK1-pY15

levels postirradiation were increased in the BECN1-WT
cells, and this effect is attributed to inactivation of the
phosphatase CDC25C21. However, the expression of
CDK1-pY15 in BECN1-KO cells was barely changed after
irradiation, which further indicated that CDK1 depho-
sphorylation in response to radiation is associated with
BECN1. As shown in Fig. 5b, the interaction between
CDK1 and CDC25C was sharply decreased in WT cells
following irradiation, which suggested the dissociation of
these two proteins. However, the cotreatment of the cells
with the autophagy inhibitor 3-MA abrogated the
radiation-induced dissociation of CDC25C from CDK1
(Fig. 6b). The interaction of CDK1 with CDC25C was
detected in both BECN1-KO and BECN1-WT cells under
normal growth conditions. CDC25C rapidly dissociated
from CDK1 in BECN1-WT cells within 1 and 2 h after 4-
Gy irradiation, whereas the interaction of CDC25C with
CDK1 was not disrupted in the irradiated BECN1-KO

cells (Fig. 6c), which is consistent with the effect of 3-MA
treatment (Fig. 5b). Importantly, the restoration of
BECN1 expression in BECN1-KO cells through
adenovirus-mediated transfection of a BECN1-expressing
vector resulted in the irradiation-induced dissociation of
CDC25C from CDK1 (Fig. 6d). In addition, CDK1 CoIP
results indicated that the interaction between CDK1 and
WEE1 increased after 4-Gy irradiation in both BECN1-
KO and BECN1-WT cells, which indicated that BECN1
does not influence the interaction between CKD1 and
WEE1 (Fig. 6c, d).
As shown in Fig. 7a, increased levels of BECN1 in the

nucleus and CDC25C in the cytoplasm were detected in
BECN1-WT cells following irradiation. However, in
BECN1-KO cells, the radiation-induced cytoplasmic
translocation of CDC25C was largely blocked. Immuno-
fluorescence staining also demonstrated that the cyto-
plasmic translocation of CDC25C was blocked in
irradiated BECN1-KO cells and could be restored fol-
lowing transfection of an exogenous BECN1-expressing
vector (Fig. 7b, c). These results are consistent with the
effects of BECN1 on the interaction between

CDC25C

and CDK1 and the dephosphorylation of CDK1 (Fig. 6).
The immunofluorescence staining assay results clearly
showed that the radiation-induced nuclear translocation
of BECN1 was largely attenuated by 3-MA (Fig. 7d). In
addition, a western blot analysis indicated that irradiation
increased the nuclear translocation of BECN1, and this
effect was reduced by treatment with 3-MA (Fig. 7e).
Moreover, the suppression of ATG5 and ATG7 expres-
sion by specific siRNAs inhibited radiation-induced
autophagy, also prevented the radiation-induced nuclear
translocation of BECN1 (Fig. 7f). These data demonstrate
the following: (i) CDK1 interacts with WEE1 and
CDC25C to form multi-protein complexes in BECN1-
expressing MDA-MB-231 cells. (ii) BECN1 deficiency
inhibition prevents the translocation of CDC25C from the
nucleus to the cytoplasm. (iii) Consequently, CDK1 is
found in a state of dephosphorylation in BECN1-deficient
cells, leading to inactivation of the G2/M checkpoint and
cell-cycle progression from the G2 to the M phase without
arrest, even under the stress induced by radiation injury.
We then wondered whether the dissociation of

CDC25C from CDK1 and its translocation from the
nucleus to cytoplasm are directly mediated by BECN1. As
shown in Fig. 8a, there exists interactions among BECN1,
CDC25C, and CHK2, and these interactions increased
following irradiation. We also observed an interaction
between BECN1 and BCL2, and this interaction was
weakened after irradiation. We did not detect any inter-
actions between BECN1 and CHK1 or among CDK1,
CCNB1, and WEE1 (Fig. 8a). As shown in Fig. 8b, the
interaction of BECN1 with CDC25C and CHK2 only
occurred in the nucleus, and this interaction increased

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following irradiation. To determine the domain(s) of
BECN1 that mediate its interactions with CDC25C and
CHK2, we generated a series of truncated BECN1 mutants
(Fig. 8c). As shown in Fig. 8d, the BECN1-B mutant, in
which amino acids 89–155 were deleted, was no longer
able to interact with CHK2, and the BECN1-C mutant, in
which amino acids 151–224 were deleted, was no longer
able to interact with CDC25C.

As shown in Fig. 8e, the interaction between CDC25C
and CHK2 was strengthened in BECN1-WT cells fol-
lowing irradiation. However, in BECN1-KO cells, the
radiation-enhanced interaction between CDC25C and
CHK2 was only observed after transfection of a vector
directing the expression of full-length BECN1 or the
BECN1-D or BECN1-E mutants. The BECN1-B and
BECN1-C mutants did not interact with CHK2 or

Fig. 6 BECN1 deficiency and autophagy inhibition inhibits the dissociation of CDC25C from the CDK1 complex after irradiation. a The levels
of phosphorylated CDK1-pY15 protein in BECN1-KO and BECN1-WT MDA-MB-231 cells were detected 1 and 2 h after 4-Gy irradiation by western blot
analysis. β-actin served as the internal loading control. b Effects of 3-MA treatment on the protein–protein interactions of the CDK1 complex in MDA-
MB-231 cells after irradiation. Cell lysates were collected from DMSO-treated MDA-MB-231 cells and 3-MA-treated cells at 2 h after 4-Gy irradiation,
and immunoprecipitates were prepared with anti-CDK1 or anti-IgG antibodies. Western blot analysis was performed using anti-CDK1, anti-CDC25C,
and anti-WEEl antibodies. β-actin was used as the internal loading control. c Effects of BECN1 deficiency on the protein–protein interactions of the
CDK1 complex after irradiation. Cell lysates were collected from BECN1-WT and BECN1-KO MDA-MB-231 cells at the indicated timepoints after 4-Gy
irradiation, and immunoprecipitates were prepared with anti-CDK1 or anti-IgG antibodies. Western blotting analysis was performed using anti-CDK1,
anti-CDC25C, and anti-WEEl antibodies. β-actin was used as the internal loading control. d Effects of BECN1 deficiency and overexpression on the
protein–protein interactions of the CDK1 complex in MDA-MB-231 cells with or without 4-Gy irradiation. Cell lysates were collected from BECN1-WT
cells, BECN1-KO cells, and BECN1-KO cells transfected with exogenous BECN1 mediated by lentiviral vectors with or without 4-Gy irradiation, and
immunoprecipitates were prepared with anti-CDK1 or anti-IgG antibodies. Western blotting analysis was performed using the indicated antibodies.
β-actin was used as the internal loading control.

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10 m 10 m 10 m10 m

10 m 10 m 10 m10 m
10 m 10 m 10 m 10 m

10 m10 m 10 m 10 m

CDC25C CDC2 DAPI Merge

BECN1-WT

No IR

BECN1-KO
No IR
BECN1-WT

4Gy IR

BECN1-KO
4Gy IR

10 m 10 m 10 m 10 m 10 m 10 m

10 m 10 m 10 m 10 m 10 m 10 m
10 m 10 m 10 m 10 m 10 m 10 m
10 m 10 m 10 m 10 m 10 m 10 m
CDC25C

BECN1 -WT BECN1 -KO
BECN1 -KO

+ BECN1

Irradiation + + +

CDK1

DAPI

Merge
10 m 10 m 10 m10 m
10 m10 m 10 m 10 m
10 m10 m 10 m 10 m

AM3OSMD

Irradiation ++

BECN1
DAPI
Merge

Fig. 7 (See legend on next page.)

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CDC25C. These results indicate that the interaction/
phosphorylation of CDC25C with/by CHK2 in response
to IR is mediated by BECN1.
Finally, we investigated the effects of BECN1 on the

assembly/interaction of the G2/M checkpoint complex
CHK2/CDC25C/CDK1/CCNB1 and its association with
CHK2 kinase activity and the phosphorylation of
CDC25C/S216 in response to IR (Fig. 8f). An HA-tagged
CHK2 immunoprecipitation (IP:HA) assay showed that
the interaction between exogenous HA-CHK2 and Flag-
CDC25C BECN1-WT cells increased following irradia-
tion. The interaction between HA-CHK2 and Flag-
CDC25C in BECN1-KO cells was lower than that in
WT cells, regardless of irradiation. However, restoration
of the expression of BECN1 allowed the radiation-induced
interaction of CHK2 and CDC25C in BECN1-KO cells.
A Flag-tagged CDC25C immunoprecipitation assay (IP:

Flag) showed that the interaction between exogenous HA-
tagged CHK2 and Flag-tagged CDC25C in BECN1-WT
cells was also enhanced following IR, whereas the inter-
action of Flag-tagged CDC25C with endogenous CDK1 in
these cells decreased following irradiation (Fig. 8f). Simi-
larly, in BECN1-KO cells, irradiation weakened the
interaction of Flag-CDC25C with HA-CHK2 but did not
decrease the interaction of Flag-CDC25C with CDK1. The
phosphorylation-mimic mutant CDC25/S216E was no
longer able to interact with CDK1. The restoration of
BECN1 expression increased the interaction of CDC25C
with CHK2 in BECN1-KO cells and decreased the inter-
action of CDC25C with CDK1 to a level similar to that
observed in BECN1-WT cells in response to radiation. A
high level of interaction was found between CDK1 and
CCNB1 in BECN1-KO cells, and this level decreased
following transfection of the phosphorylation-mimic
CDC25C-S216E expression vector. Restoration of
BECN1 expression decreased the interaction of CDK1
with CCNB1 in irradiated BECN1-KO cells.

The BECN1 and CDK1 expression levels are increased in
breast cancer tissue samples
To determine whether the expression of BECN1 and

CDK1 are altered in breast cancer tissues, gene expression

data from the Gene Expression Omnibus (GEO) database
(accession numbers GSE81838 and GSE65194) and the
breast cancer patient dataset from the Cancer Genome
Atlas (TCGA) were analyzed22. As shown in Supple-
mentary Fig. 6a, 93 genes overlapped among the three
datasetsGSE65194, GSE81838, and TCGA datasets, of
which BECN1 and CDK1 were both upregulated in breast
cancer tissue compared with normal tissue. Supplemen-
tary Fig. 6b presents the relative expression levels of
several essential autophagy-related genes, including
BECN1 and G2/M-regulated genes, such as CDK1,
CDC25C, and CHK1, in breast cancer and normal tissues
in the TCGA dataset. We also found that both BECN1
and CDK1 are upregulated in breast cancer tissue com-
pared with normal tissue (Supplementary Fig. 6c). Several
essential autophagy-related and G2/M-regulating genes,
including BECN1, CDK1, and CDC25C, are coexpressed;
in particular, CDK1 is associated with both autophagy-
related and G2/M-regulating genes (Supplementary Fig. 6d).
Therefore, BECN1 was translocated into the nucleus

following IR, where it mediated the interaction of
CDC25C with CHK2, prompted the phosphorylation of
CDC25C and its dissociation from CDK1 and thus
resulted in the inactivation of the CDK1/CCNB1 complex
and arrest at the G2/M transition in the cell cycle, leading
the CDK1 overexpression to promote the radiation-
induced EMT (Supplementary Fig. 7).

Discussion
Autophagy and cell-cycle arrest are two critical cellular

responses to IR, and autophagy is induced even as part of
the radiation-induced bystander effect23,24. Because
initiation is potentiated by the impairment of autophagy
through the disruption of core autophagy genes and
autophagy-defective tumor cells also display a dysregu-
lated cell cycle25, we, in contrast to previous studies, used
the autophagy inhibitor 3-MA and BECN1-KO cancer
cells to directly determine the role of autophagy in G2/M
arrest. The results of our study suggest that BECN1
deficiency enhances cellular sensitivity to IR, induces
escape from the G2/M checkpoint after irradiation and
promotes the G2/M transition without arrest. These two

(see figure on previous page)
Fig. 7 The radiation-induced cytoplasmic translocation of CDC25C is blocked under BECN1-deficient conditions. a The levels of CDC25C and
BECN1 proteins in the cytoplasm and nucleus, respectively, in BECN1-KO and BECN1-WT MDA-MB-231 cells after 4-Gy irradiation were detected by
Western blot analysis. β-actin served as the internal loading control. Tubulin and LAMIN A/C proteins were detected as the control cytoplasmic and
nuclear proteins, respectively. Exogenous BECN1-expressing vectors were transfected into BECN1-KO cells for the rescue experiment. b CDC25C and CDK1
in BECN1-WT and BECN1-KO MDA-MB-231 cells with or without 4-Gy γ-ray-irradiation were detected by immunofluorescence confocal microscopy(bars:
×20 magnification). c CDC25C and CDK1 in BECN1-WT and BECN1-KO MDA-MB-231 cells with or without 4-Gy γ-ray-irradiation were detected by
immunofluorescence confocal microscopy. Exogenous BECN1-expressing vectors were transfected into BECN1-KO cells for the rescue experiment(bars:
×20 magnification). d. The BECN1 levels after 4-Gy γ-ray-irradiation with or without 3-MA treatment were assayed by immunofluorescence microscopy
(bars: ×20 magnification). e The levels of BECN1, tubulin and LAMIN A/C proteins in the cytoplasm and nucleusafter 4-Gy irradiation with or without
cotreatment with 3-MA were detected by Western blot analysis. β-actin served as the internal loading control. f Effects of siRNA-mediated knockdown of
ATG5 and ATG7 on the radiation-induced nuclear translocation of BECN1.

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events [(1) the suppression of autophagy post-IR pro-
motes cell death and suppresses proliferation and (2)
the suppression of autophagy induces escape from the
G2/M checkpoint and promotes the G2/M transition]
appear to be but are not actually contradictory. On the
one hand, the inhibition of autophagy can promote the

G2/M transition in unrepaired cells, and on the other
hand, mitotic arrest can be induced in cells damaged by
radiation. Moreover, the cells that escape G2/M arrest
enter the M phase without undergoing adequate repair,
which will likely result in mitotic catastrophic cell
death26.

Fig. 8 BECN1 mediates the interaction of CDC25C with CHK2. a The interactions of BECN1 with CDC25C, CHK2, CHK1, 13-3-3α, WEE1, CDK1,
CCNB1, and BCL2in MDA-MB-231 cells with or without 4-Gy irradiation were assayed, and immunoprecipitates were prepared with anti-BECN1 or
anti-IgG antibodies. Western blot analyses were performed using the indicated antibodies. b The interactions of BECN1 with CDC25C and CHK2 in
either the cytoplasm or nucleusof MDA-MB-231 cells with or without 4-Gy irradiation were assayed, and immunoprecipitates were prepared with
anti-BECN1, anti-CHK2, anti-CDC25C, or anti-IgG antibodies. Western blot analysis was performed using anti-CDC25C and anti-CHK2 antibodies.
c BECN1-truncated mutants were constructed according to the listed illustrator. d Flag-BECN1, flag-BECN1-A, flag-BECN1-B, flag-BECN1-C, flag-BECN1-
D, flag-BECN1-E, and empty vector were overexpressed in MDA-MB-231 cells. Immunoprecipitation was performed using a flag antibody. e Flag-
BECN1, flag-BECN1-A, flag-BECN1-B, flag-BECN1-C, flag-BECN1-D, flag-BECN1-E, and empty vector were overexpressed in BECN1-KO MDA-MB-231
cells. Immunoprecipitation was performed using anti-CDC25 antibody, and western blot analysis was performed using anti-CDC25C and anti-CHK2
antibodies. f The cells were transfected with the indicated Flag- or HA-tagged expression vectors, and the immunoprecipitations were performed
using anti-HA, anti-Flag, or anti-IgG antibodies. Western blot analyses were performed using anti-Flag, anti-HA, anti-CDK1, and anti-CCNB1 antibodies.
β-actin served as the internal loading control.

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BECN1 is a key protein in the regulation of autophagy
through the activation of VPS3427. Xiao et al. demon-
strated that macroautophagy is regulated by the cell-cycle
protein Sdk1, which impairs the interaction of BECN1
with VPS3428. CDK1 is an important player in macro-
autophagy suppression during the M phase. CDK1 can
directly phosphorylate VPS34, which prevents formation
of the BECN1-VPS34 complex and leads to decreased
autophagy in M-phase cells29. In contrast, CDK inhibitors
stimulate autophagy by releasing BECN1, which results in
the promotion of tumor growth30. Our study revealed the
involvement of autophagy in the regulation of the G2/M
checkpoint. Autophagy dysregulation can disrupt arrest at
the G2/M transition following irradiation, primarily by
affecting the dissociation of CDC25C from CDK1 and
decreasing the phosphorylation of ATM, CHK1, CHK2,
and CDK1 in the absence of BECN1. To the best of our
knowledge, this study provides the first demonstration
that autophagy and the G2/M transition are associated
with the dephosphorylation or phosphorylation of various
G2/M-regulating proteins.
Progression from the G2 to the M phase is driven by

activation of the CDK1/CCNB1 complex31. Our study
provides further evidence clarifying the mechanism
through which autophagy regulates the G2/M transition.
As indicated by previous studies, basal autophagy occurs
at different phases of the cell cycle in response to envir-
onmental genotoxins such as IR32–34. Li et al. demon-
strated that increasing the expression of ATG5 inATG5-
deficient cells rescues the cells from G2/M arrest, whereas
expression of the ATG5-K130R mutant does not produce
this effect; the rescue from G2/M arrest coincides with
increased levels of pCDK1 and CDKN1A/p2135. Jia et al.
demonstrated that autophagy regulates the negative cell-
cycle regulator CDKN1B in naïve T cells and reduces its
abundance in T cells after their activation to enter the cell
cycle36. Liu et al. reported that MJ-66, a tumor growth
inhibitor, induces glioma cell-cycle arrest at the G2/M
transition and increases the expression of PDK1 and
CDK131. In the present study, we used CoIP assays to
reveal that in BECN1-WT cells, CDC25C dissociates from
the CDK1 complex after irradiation, which results in
increased phosphorylation of CDK1 by WEE1 and thus
arrest at the G2/M transition in the cell cycle. However, in
BECN1-deficient cells, the dissociation of CDC25C from
CDK1 is attenuated, which results in CDK1 depho-
sphorylation and promotion of the G2/M transition even
under the stress induced by radiation injury. Xu et al.
reported that following B19V infection, activated ATR
phosphorylates CDC25C, which in turn inactivates the
CCNB1-CDK1 complex37. Jaceosidin, isolated from
Japanese mugwort, induces arrest at the G2/M transition
in the cell cycle through inactivation of the CDC25C-
CDK1 complex38. Vera et al. reported that both CDK1

and WEE1 are mediators of G2/M arrest
39. Our study

showed that BECN1 interacts with CDC25C and CHK2
using different domains. BECN1 deficiency decreased the
interaction of CDC25C with CHK2 and stabilized the
interaction of CDC25C with CDK1 even in the presence
of IR. Our study elucidated that under IR stress, autop-
hagy promotes G2/M arrest by targeting the formation of
the CDC25C-CHK2, CDK1-CDC25C, and CDK1-WEE1
complexes.
In conclusion, our study demonstrates that BECN1

plays a role in promotion of radiation-induced G2/M
arrest (Figs. 7g and 8). However, whether such functions
of BECN1 in G2/M arrest is dependent or independent on
its autophagy-related roles is necessary to further identify.

Materials and methods
Reagents and antibodies
The reagents used in this study were as follows: lipo-

somes were purchased from Invitrogen (Carlsbad, CA,
USA); glycine, lauryl sodium sulfate, tetra-
methylethylenediamine, TRIzol, and tris(hydroxymethyl)
aminomethane were purchased from Amresco (Solon,
OH, USA); acrylic amide was purchased from Merck
(Darmstadt, Germany); bovine serum albumin (BSA) was
purchased from Roche (Basel, Switzerland); fluorescent
protein solutions were purchased from Pierce (Rockford,
IL, USA); ammonium peroxydisulfate, dimethyl sulfoxide
(DMSO), N, N’-methylenebisacrylamide, and puromycin
were purchased from Sigma (St. Louis, MO, USA); tryp-
sin, Dulbecco’s modified Eagle’s medium (DMEM) with
high glucose, and fetal bovine serum were purchased from
HyClone (Logan, UT, USA); PrimeSTAR DNA poly-
merase and T4 DNA ligase were purchased from TaKaRa
(Tokyo, Japan); GAPDH, P62, CHK1, CHK2, MYTl,
WEEl, CDC25, CDC25C, ATM, CDK1, LC3, p68-CHK1,
p216-CDC25C, p15-CDK1, p1981-ATM, SQSTM1/P62,
pCHK2, pCHK1, CDC, pCDC25C, and MPM2 antibodies
were purchased from Santa Cruz Biotechnology (Dallas,
TX, USA), Cell Signaling Technology (Danvers, MA,
USA), or Millipore/Upstate(NY, USA); and the pLKO.1
plasmid was purchased from Sigma (Darmstadt,
Germany).

Cell lines, mouse model, and irradiation conditions
Human A549 cells and the human TNBC MDA-MB-

231 cell line were purchased from the American Type
Culture Collection (ATCC, Manassas, VA, USA). The
cells were cultured in DMEM (a high-glucose medium
containing penicillin, streptomycin, and 10% FBS) and
incubated at 37 °C under 5% CO2. The BECN1-KO
MDA-MB-231 cell line was successfully established pre-
viously in our laboratory using the CRISPR/Cas9 system
according to the free online design tool (http://crispr.mit.
edu/) 7. The cells were irradiated with 60Co γ-rays at a

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http://crispr.mit.edu/

http://crispr.mit.edu/

dose rate of 127.15 cGy/min at room temperature at the
Institute of Radiation Medicine, Academy of Military
Medical Sciences (Beijing, China). Twenty C57BL/6 male
mice (6–7 week old) were divided into two groups ran-
domly, 20 Gy γ-rays were used to induce the lung fibrosis
model. Mice were anesthetized with intraperitoneal
sodium pentobarbital (80 mg/kg). Lung tissues were col-
lected for further assay. The animal study has been
approved by ethics committee of Xiangya School of Public
Health, Central South University.

Cell transfection
The cells were passaged the day before transfection.

After the cells were grown to 60% density, BECN1 siRNA
knockdown was conducted through the transient trans-
fection of validated BECN1 siRNA (sense, 5’-GCUGCC
GUUAUACUGUUCUTT-3’, antisense, 5’-AGAACAGUA
UAACGGCAGCTT-3’) using Lipofectamine 2000
(Thermo Fisher Scientific, Waltham, MA, USA) following
the manufacturer’s instructions. Scrambled siRNA was used
as the negative control. Forty-eight hours after transfection,
the cells were collected for further experiments.

Cell proliferation and colony formation assays
Cell proliferation was assessed using the CCK-8 col-

orimetric assay (Dojindo Molecular Technologies,
Kumamoto, Japan). The cells were divided into the fol-
lowing treatment groups: 0 Gy, 3-MA, 4 Gy + 3-MA,
3-MA at 0 h, 4 Gy, and 4 Gy + 3-MA. The cells were then
cultured in a 96-well plate at a density of 4 × 103 cells/well
at 37 °C in the presence of 5% CO2for 12, 24, 48, and 72 h,
and the level of cell proliferation was then assessed. The
optical density (OD) of each well at 450 nm was read
using a Multiskan GO microplate reader (Thermo Fisher
Scientific). Each experiment was performed in triplicate.
The colony formation ability was used to assess the cell

survival percentage. After treatment with 3-MA, 2 Gy,
2 Gy + 3-MA, 4 Gy, or 4 Gy + 3-MA, the cells were see-
ded into 60-mm culture dishes at a density of 1000 cells/
dish. After 2 weeks, the cells were stained with crystal
violet. The number of microscopic colonies with more
than 50 cells was counted. The cell survival ratio based on
the number of colony-forming irradiated cells compared
with that of the control cells was calculated.

Western blotting analysis
A Western blotting analysis was performed for the

detection of proteins or phosphorylated proteins. Briefly, the
samples were treated with lysis buffer, subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE), and transferred to polyvinylidene fluoride mem-
branes. After the membranes were blocked with 5% nonfat
milk in Tris-buffered saline containing Tween-20 (TBST)
for 2 h, the membranes were incubated with primary and

secondary antibodies overnight at 4 °C. An ImageQuant
LAS500 system (Molecular Dynamics, Sunnyvale, CA, USA)
was used to visualize the bands. Details of the Western blot
analysis can be found in our previous publications34,40–43.

Apoptosis detection
In this study, apoptosis was assessed using a Fluorescein

Isothiocyanate (FITC)-Annexin V Apoptosis Detection
Kit (BD Pharmingen, San Diego, CA, USA) following the
manufacturer’s instructions. The cells were treated with
or without 3-MA, subjected to 4-Gy irradiation and har-
vested at 24, 48, or 72 h after irradiation. The cells were
washed twice with 3 ml of phosphate-buffered saline
(PBS), and RNase A was then added. The cells were
centrifuged, resuspended in PBS and transferred to clean
Eppendorf tubes, and 100 µl of 1× Annexin V-binding
solution was then added to the cells to form a suspension
of 1 × 10 cells/ml. Subsequently, 5 μl of FITC-conjugated
Annexin V and propidium iodide (PI) solution (40 μg/ml
PI and 0.1% Triton X-100 in PBS buffer) was added. The
cells were then incubated for 15 min at room temperature
in the dark, and 400 μl of 1× binding buffer was then
added. The cells were then analyzed by flow cytometry44.

Cell-cycle analysis and G2/M arrest assay
The cells were seeded into 35-mm culture dishes at a

density of 70–80% per dish. The cells were treated or not
treated with 3-MA, subjected to irradiation 2 h after the
pretreatment, and harvested at the indicated timepoints
(0, 2, 4, 6, 8, or 12 h) after irradiation. After the medium
was removed, the cells were treated with RNase A (62 μg/
ml) and incubated at 37 °C for 30 min. The cells were
stained with PI solution, and the cell-cycle distribution
was analyzed by flow cytometry.
For the comparison of G2/M arrest between radiation-

induced, 3-MA-treated, and BECN1-knockout cells, the
mitotic cells were counted using a FACSCalibur flow
cytometer (BD Pharmingen). Cells that were treated or
not treated with 3-MA or in which BECN1 was knocked
out were subjected or not subjected to irradiation and
harvested at the indicated timepoints (0, 2, 4, 6, 8, or 12 h)
postirradiation. After irradiation, PBS was added to sus-
pend the cells; the cells were then centrifuged at
2000 rpm, and 0.25% Triton X-100 was added to induce
membrane rupture. After the addition of 40 μl of 1% BSA
containing anti-Ser10-phosphorylated histone H3 anti-
body, the cells were incubated for 50 min at room tem-
perature, and 80 μl of 1% BSA containing a FITC-tagged
secondary antibody was then added. The cells were then
incubated for 30 min and stained with PI solution (20 μg/
ml) for 10–30 min at room temperature, and the mitosis
stage was analyzed by flow cytometry. Two‐dimensional
dot plots were generated using ModFit LT software
(Verity Software House, Inc., Topsham, ME, USA).

Huang et al. Cell Death Discovery (2020) 6:70 Page 15 of 17

Official journal of the Cell Death Differentiation Association

Coimmunoprecipitation (CoIP)
For the CoIP assay, normal MDA-MB-231 cells and

BECN1-KO MDA-MB-231 cells that were subjected or
not subjected to 4-Gy irradiation were washed, harvested,
and lysed with PBS buffer containing 50 nM Tris-base,
1 mM EDTA, 1% NP-40, and 1× protease inhibitor
cocktail. The lysates were centrifuged, and the super-
natant was collected for the CoIP assay using the Pierce
Classic IP Kit (Thermo Scientific). CDK1 antibody was
used to form immunocomplexes with CDC25C and
WEEl, and WEEl antibody was used to form immuno-
complexes with CDK1. These immunocomplexes were
isolated by 8% SDS-PAGE, washed, and eluted, and the
protein interactions were detected by western blotting.

Immunofluorescence staining and laser confocal
microscopy
Autophagy and subcellular protein localization were

analyzed by immunofluorescence staining and laser con-
focal microscopy observations. Autophagy was analyzed by
quantifying the formation of puncta of the autophagy bio-
marker GFP-LC3 by immunofluorescence staining and
laser confocal microscopy. HeLa cells were seeded in glass
chamber slides. After 12 h, the cells were transfected with
GFP-LC3 using Lipofectamine 2000. Four hours after
transfection, the cells were irradiated with 4 Gy and treated
with 0.25% Triton X-100, and the nuclei were stained with
DAPI for visualization. The GFP-LC3 puncta per cell were
counted. The samples were observed using an LSM 510
laser-scanning confocal microscope (Zeiss, Germany).

Coexpression network construction
To construct a coexpression network between

autophagy-mediated genes and G2/M checkpoint genes,
we analyzed the differential expression of autophagy-
mediated mRNAs and G2/M checkpoint mRNAs (Sup-
plementary Table 1) using gene expression data obtained
from the GEO database (Accession Nos. GSE65194 and
GSE81838) (https://www.ncbi.nlm.nih.gov/) and the
breast cancer dataset from the TCGA (https://
cancergenome.nih.gov/). The ranks of essential autop-
hagy genes and G2/M checkpoint genes were determined
by the absolute differences in their expression between
the control and breast cancer groups. For each pair ana-
lyzed, we used the Pearson correlation test to detect sig-
nificant correlations. Only Pearson correlation
coefficients ≥0.9 (p < 0.01) were used to construct the network and generate visual representations.

Acknowledgements
We thank Prof. Yungui Yang (Beijing Institute of Genomics, CAS, China) for the
helpful suggestions and comments on this manuscript. This study was
supported by grants from the National Natural Science Foundation of China
(Grant Nos. 31530085, 31870847, U1803124, and 81842033), and the National

Key Basic Research Program (973 Program) of MOST, China (Grant No.
2015CB910601).

Author details
1Department of Occupational and Environmental Health, Xiangya School of
Public Health, Central South University, 410078 Changsha, Hunan Province,
China. 2Department of Radiation Biology, Beijing Key Laboratory for
Radiobiology, Beijing Institute of Radiation Medicine, AMMS, 100850 Beijing,
China. 3Institute for Chemical Carcinogenesis, State Key Laboratory of
Respiratory, School of Public Health, Guangzhou Medical University, 511436
Guangzhou, P. R. China

Author contributions
R.H., H.N., and Y.Z. are coming from Department of Occupational and
Environmental Health, Xiangya School of Public Health, Central South
University, Changsha, Hunan Province 410078, China. S.G., Y.H., H.G., X.L., S.Y.,
and P-K.Z. are coming from Department of Radiation Biology, Beijing Key
Laboratory for Radiobiology, Beijing Institute of Radiation Medicine, AMMS,
Beijing 100850, China.

Conflict of interest
The authors declare that they have no conflict of interest.

Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.

The online version of this article (https://doi.org/10.1038/s41420-020-00301-2)
contains supplementary material, which is available to authorized users.

Received: 14 April 2020 Revised: 21 May 2020 Accepted: 28 May 2020

References
1. Ye, H. et al. Chloroquine, an autophagy inhibitor, potentiates the radio-

sensitivity of glioma initiating cells by inhibiting autophagy and activating
apoptosis. BMC Neurol. 16, 178 (2016).

2. Yu, L. et al. DAB2IP regulates autophagy in prostate cancer in response to
combined treatment of radiation and a DNA-PKcs inhibitor. Neoplasia 14,
1203–1212 (2012).

3. Kuwahara, Y. et al. Association between radiation-induced cell death and
clinically relevant radioresistance. Histochem. Cell Biol. 150, 649–659 (2018).

4. Taylor, M. A., Das, B. C. & Ray, S. K. Targeting autophagy for combating
chemoresistance and radioresistance in glioblastoma. Apoptosis Int. J. Program.
Cell death 23, 563–575 (2018).

5. Chen, C., Wang, K., Wang, Q. & Wang, X. LncRNA HULC mediates radio-
resistance via autophagy in prostate cancer cells. Braz. J. Med. Biol. Res. 51,
e7080 (2018).

6. Zou, Y. M. et al. Hypoxia-induced autophagy contributes to radioresistance via
c-Jun-mediated Beclin1 expression in lung cancer cells. J. Huazhong Univ. Sci.
Technol. Med. Sci. 34, 761–767 (2014).

7. Wu, C. L. et al. BECN1-knockout impairs tumor growth, migration and invasion
by suppressing the cell cycle and partially suppressing the epithelial-
mesenchymal transition of human triple-negative breast cancer cells. Int. J.
Oncol. 53, 1301–1312 (2018).

8. Li, B. et al. Tumor-initiating cells contribute to radiation resistance in primary
human renal clear cell carcinomas by activating the DNA damage checkpoint
response. Oncol. Lett. 14, 3261–3267 (2017).

9. Carruthers, R. et al. Abrogation of radioresistance in glioblastoma stem-like
cells by inhibition of ATM kinase. Mol. Oncol. 9, 192–203 (2015).

10. Mei, Y., Glover, K., Su, M. & Sinha, S. C. Conformational flexibility of BECN1:
essential to its key role in autophagy and beyond. Protein Sci. 25, 1767–1785
(2016).

11. Chen, K. et al. Artesunate induces G2/M cell cycle arrest through autophagy
induction in breast cancer cells. AntiCancer Drugs 25, 652–662 (2014).

12. Menon, M. B. & Dhamija, S. Beclin 1 phosphorylation—at the center of
autophagy regularization. Front. Cell Dev. Biol. 6, 137 (2018).

Huang et al. Cell Death Discovery (2020) 6:70 Page 16 of 17

Official journal of the Cell Death Differentiation Association

https://www.ncbi.nlm.nih.gov/

https://cancergenome.nih.gov/

https://cancergenome.nih.gov/

https://doi.org/10.1038/s41420-020-00301-2

13. Zhong, R. et al. Deoxycytidine kinase participates in the regulation of radiation-
induced autophagy and apoptosis in breast cancer cells. Int. J. Oncol. 52,
1000–1010 (2018).

14. Vicinanza, M., Puri, C. & Rubinsztein, D. C. Coincidence detection of RAB11A
and PI(3)P by WIPI2 directs autophagosome formation. Oncotarget 10,
2579–2580 (2019).

15. Morishita, H., Kaizuka, T., Hama, Y. & Mizushima, N. A new probe to measure
autophagic flux in vitro and in vivo. Autophagy 13, 757–758 (2017).

16. Xu, J. et al. Omi/HtrA2 participates in age-related autophagic deficiency in rat
liver. Aging Dis. 9, 1031–1042 (2018).

17. Moniz, L. S. & Stambolic, V. Nek10 mediates G2/M cell cycle arrest and MEK
autoactivation in response to UV irradiation. Mol. Cell. Biol. 31, 30–42 (2011).

18. Huang, B. et al. DNA-PKcs associates with PLK1 and is involved in proper chro-
mosome segregation and cytokinesis. J. Cell. Biochem. 115, 1077–1088 (2014).

19. Wang, J. et al. Tetrandrine enhances radiosensitivity through the CDC25C/
CDK1/cyclin B1 pathway in nasopharyngeal carcinoma cells. Cell Cycle 17,
671–680 (2018).

20. Fisher, D., Krasinska, L., Coudreuse, D. & Novak, B. Phosphorylation network
dynamics in the control of cell cycle transitions. J. Cell Sci. 125, 4703–4711
(2012).

21. Forester, C. M., Maddox, J., Louis, J. V., Goris, J. & Virshup, D. M. Control of
mitotic exit by PP2A regulation of Cdc25C and Cdk1. Proc. Natl Acad. Sci. USA
104, 19867–19872 (2007).

22. Lehmann, B. D. et al. Refinement of triple-negative breast cancer molecular
subtypes: implications for neoadjuvant chemotherapy selection. PloS ONE 11,
e0157368 (2016).

23. Song, M. et al. Bystander autophagy mediated by radiation-induced exosomal
miR-7-5p in non-targeted human bronchial epithelial cells. Sci. Rep. 6, 30165
(2016).

24. Cai, S. et al. Exosomal miR-7 mediates bystander autophagy in lung after focal
brain irradiation in mice. Int J. Biol. Sci. 13, 1287–1296 (2017).

25. Karantza-Wadsworth, V. et al. Autophagy mitigates metabolic stress and
genome damage in mammary tumorigenesis. Genes Dev. 21, 1621–1635
(2007).

26. Shang, Z. F. et al. Inactivation of DNA-dependent protein kinase leads to
spindle disruption and mitotic catastrophe with attenuated Chk2 phosphor-
ylation in response to DNA damage. Cancer Res. 70, 3657–3666 (2010).

27. Maejima, Y., Isobe, M. & Sadoshima, J. Regulation of autophagy by Beclin 1 in
the heart. J. Mol. Cell. Cardiol. 95, 19–25 (2016).

28. Xiao, J. et al. FBXL20-mediated Vps34 ubiquitination as a p53 controlled
checkpoint in regulating autophagy and receptor degradation. Genes Dev. 29,
184–196 (2015).

29. Eskelinen, E. L. et al. Inhibition of autophagy in mitotic animal cells. Traffic 3,
878–893 (2002).

30. Pimkina, J., Humbey, O., Zilfou, J. T., Jarnik, M. & Murphy, M. E. ARF induces
autophagy by virtue of interaction with Bcl-xl. J. Biol. Chem. 284, 2803–2810
(2009).

31. Liu, W. T. et al. MJ-66 induces malignant glioma cells G2/M phase arrest and
mitotic catastrophe through regulation of cyclin B1/Cdk1 complex. Neuro-
pharmacology 86, 219–227 (2014).

32. Huang, R. & Zhou, P. Double-edged effects of noncoding RNAs in responses
to environmental genotoxic insults: Perspectives with regards to molecule-
ecology network. Environ. Pollut. 247, 64–71 (2019).

33. Zhou, P. K. & Huang, R. X. Targeting of the respiratory chain by toxicants:
beyond the toxicities to mitochondrial morphology. Toxicol. Res. (Camb.) 7,
1008–1011 (2018).

34. Mo, L. J. et al. Exosome-packaged miR-1246 contributes to bystander DNA
damage by targeting LIG4. Br. J. Cancer 119, 492–502 (2018).

35. Li, H. et al. Atg5-mediated autophagy deficiency in proximal tubules promotes
cell cycle G2/M arrest and renal fibrosis. Autophagy 12, 1472–1486 (2016).

36. Jia, W. et al. Autophagy regulates T lymphocyte proliferation through selective
degradation of the cell-cycle inhibitor CDKN1B/p27Kip1. Autophagy 11,
2335–2345 (2015).

37. Xu, P. et al. Parvovirus B19 NS1 protein induces cell cycle arrest at G2-phase by
activating the ATR-CDC25C-CDK1 pathway. PLoS Pathog. 13, e1006266 (2017).

38. Lee, J. G. et al. Jaceosidin, isolated from dietary mugwort (Artemisia princeps),
induces G2/M cell cycle arrest by inactivating cdc25C-cdc2 via ATM-Chk1/2
activation. Food Chem. Toxicol. 55, 214–221 (2013).

39. Vera, J. et al. Chk1 and Wee1 control genotoxic-stress induced G2-M arrest in
melanoma cells. Cell. Signal. 27, 951–960 (2015).

40. Qin, J. et al. LncRNA Uc.173 is a key molecule for the regulation of lead-
induced renal tubular epithelial cell apoptosis. Biomed. Pharmacother. 100,
101–107 (2018).

41. Qin, J. et al. LncRNA MIR31HG overexpression serves as poor prognostic
biomarker and promotes cells proliferation in lung adenocarcinoma. Biomed.
Pharmacother. 99, 363–368 (2018).

42. Shen, L. et al. LncRNA lnc-RI regulates homologous recombination repair of
DNA double-strand breaks by stabilizing RAD51 mRNA as a competitive
endogenous RNA. Nucleic acids Res. 46, 717–729 (2018).

43. Jin, F. et al. Inhibitory effect of uranyl nitrate on DNA double-strand break
repair by depression of a set of proteins in the homologous recombination
pathway. Toxicol. Res. (Camb.) 6, 711–718 (2017).

44. Li, M. et al. PIG3 promotes NSCLC cell mitotic progression and is associated
with poor prognosis of NSCLC patients. J. Exp. Clin. Cancer Res. 36, 39 (2017).

Huang et al. Cell Death Discovery (2020) 6:70 Page 17 of 17

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• BECN1 promotes radiation-induced G2/M arrest through regulation CDK1 activity: a potential role for autophagy in G2/M checkpoint

Introduction
Results
BECN1 protein was remarkably upregulated in A549 cells, Hela cells, and mice lung fibrosis tissues
BECN1 and its mediated autophagy were essential for cell response to radiation and lung fibrosis
G2/M checkpoint was regulated by BECN1 in response to IR
BECN1 deficiency disrupts the responses of G2/M checkpoint proteins to IR
BECN1 deficiency disrupts the dissociation of CDC25C from CDK1 following irradiation
The BECN1 and CDK1 expression levels are increased in breast cancer tissue samples
Discussion
Materials and methods
Reagents and antibodies
Cell lines, mouse model, and irradiation conditions
Cell transfection
Cell proliferation and colony formation assays
Western blotting analysis
Apoptosis detection
Cell-cycle analysis and G2/M arrest assay
Coimmunoprecipitation (CoIP)
Immunofluorescence staining and laser confocal microscopy
Coexpression network construction
Acknowledgements

BECN1 promotes radiation-induced G2/M arrest through regulation CDK1 activity: a potential role for autophagy in G2/M checkpoint. 
Huang, R., Gao, S., Han, Y. et al. 
Cell Death Discov. 6, 70 (2020). https://doi.org/10.1038/s41420-020-00301-2

Complete above citation

Molecules 2017, 22(12), 2045; https://doi.org/10.3390/molecules22122045
Introduction: Cell cycle control: DNA damage checkpoint
To highlight with arrows or boxes key points
see notes below for details

Cell cycle control: DNA damage checkpoint (modified from Aarts et al. [55]). Reproduced with permission from Nick Turner, Current Opinion in Pharmacology; published by Elsevier, 2013.
In the cell cycle, there are two checkpoint arrests that allow cells to repair damaged DNA in order to maintain genomic integrity. Many cancer cells have defective G1 checkpoint mechanisms, thus depending on the G2 checkpoint far more than normal cells. G2 checkpoint abrogation is therefore a promising concept to preferably damage cancerous cells over normal cells. The main factor influencing the decision to enter mitosis is a complex composed of Cdk1 and cyclin B. Cdk1/CycB is regulated by various feedback mechanisms, in particular inhibitory phosphorylations at Thr14 and Tyr15 of Cdk1. In fact, Cdk1/CycB activity is restricted by the balance between WEE family kinases and Cdc25 phosphatases. The WEE kinase family consists of three proteins: WEE1, PKMYT1, and the less important WEE1B. WEE1 exclusively mediates phosphorylation at Tyr15, whereas PKMYT1 is dual-specific for Tyr15 as well as Thr14. Inhibition by a small molecule inhibitor is therefore proposed to be a promising option since WEE kinases bind Cdk1, altering equilibria and thus affecting G2/M transitio
2

Intoduction: Autophagy and Beclin 1 (BECN1) phosphorylation. 
Front. Cell Dev. Biol., 12 October 2018 | https://doi.org/10.3389/fcell.2018.00137
To highlight with arrows or boxes key points
see notes below for details

 (A) The scheme depicts the phases of autophagosome assembly from initiation to nutrient recycling. The steps include the structural transformation from the pre-autophagosomal structure (PAS) to phagophore to autophagosomes, culminating in the fusion of autophagosomes with lysosomes facilitating the degradation of their contents in autolysosomes. The regulatory protein complexes involved are depicted with their components and the presence of regulatory phosphorylation event/events are shown. (B) Primary structure of BECN1 showing the BCL2/BCL-XL binding BH3 motif (residues 105–130), flexible helical domain (F, residues 141–171), and the central coiled coil domain (CCD, residues 175–265). Evolutionary conserved domain (residues 248–337) and β/α-repeated, autophagy-related (BARA) domain (265–450aa) is represented together as ECD-BARA (Mei et al., 2016). The approximate locations of pro-autophagy (green) and inhibitory (red) phosphorylation sites are shown. The contributions of the different domains to complex formation with interactors are also indicated. (C) Phosphorylation-dependent conversion of inactive BECN1 homodimer/BCL2-complex to an active PI3K-III complex is depicted. The STK4-mediated BECN1-BH3 domain phosphorylation (negative regulator of autophagy), triple phosphorylation of BCL2 which releases BCL2 from BECN1, representative phosphorylation events in the N-terminal domain (NTD) promoting BECN1-BCL2 dissociation as well as activating the PI3K-activity are presented (positive regulation).
3

Materials and Methods
Cell lines and animal models
Reagents and drugs
Major Assays
Complete this slide

Fig. 1 A-G
Assays:
A. qRT-PCR
B. qRT-PCR
C. Western blotting
D. Western blotting
E. Quantification of Fluorescence Confocal Microscopy of LC3 Puncta
F. Fluorescence Confocal Microscopy
G. Fluorescence Confocal Microscopy
Cell lines:
D-G. MDA-MB-231
Abbreviations and Treatments
3MA: ?
Wortamannin? ?

Complete each panel in Fig 1. with 1) cell lines or models- if not shown in the Fig. , 2) Key points with arrows or boxes , 3) assay used, 4) drug or other treatments if not shown in the fig and 5) abbreviations used spelled out

5

Fig. 1 A-G
Panel A.
Panel B.
Panel C.
Panel D.
Panel E.
Panel F.
Panel G.

 Fig. 1: BECN1 was overexpressed in MDA-MB-231, A549, and mouse tissues post-exposure of IR.
a. qRT-PCR was performed to detect BECN1 mRNA expression and the asterisk (*) indicates a significant increase post 4 Gy IR exposure compared to the non-IR exposure in MDA-MB-231 cells. b qRT-PCR was performed to detect BECN1 mRNA expression and the asterisk (*) indicates a significant increase post 4 Gy IR exposure compared to the non-IR exposure in A549 cells. c Western blot assay was performed to detect theATG3 and BECN1 expression in MDA-MB-231 cells (n = 5) post 4 Gy IR exposure. d The protein levels of LC3 and SQSTM1/P62 in MDA-MB-231 cells were detected by Western blotting analysis. GAPDH was used as the loading control. MDA-MB-231 cells cotreated or not cotreated with 3-MA were harvested at the indicated timepoints after 4-Gy γ-ray-irradiation. e MDA-MB-231 cells cotreated or not cotreated with 3-MA or wortamannin were harvested at the indicated timepoints after 4-Gy γ-ray-irradiation. The number of LC3 puncta per cell. was analyzed and the asterisk (*) indicates a significant change compared with control group. f Autophagic GFP-LC3 puncta in 4-Gy γ-ray-irradiated MDA-MB-231 cells cotreated with or without 3-MA were detected by fluorescence confocal microscopy(bars: ×20 magnification). g Autophagic WIPI2in 4-Gy γ-ray-irradiated MDA-MB-231 cells cotreated with or without 3-MA was detected by fluorescence confocal microscopy(bars: ×20 magnification). The data are presented as the means ± SDs from three independent experiments *p < 0.01 compared with the control group. 6 Fig. 2 A-G Complete each panel in Fig 2. with 1) cell lines or models- if not shown in the Fig. , 2) Key points with arrows or boxes , 3) assay used, 4) drug or other treatments if not shown in the fig and 5) abbreviations used spelled out 7 Panel A. Panel B. Panel C. Panel D. Panel E. Panel F. Panel G. Fig. 2 A-G Fig. 2: Involvement of autophagy in the regulation of IR-induced EMT. a. The BECN1 expression level was analyzed in 20 paired mice lung fibrosis tissues with 20 Gy radiation and their normal tissues without 20 Gy radiation by calculating the integrated IOD/area using Image-Pro Plus version 6.0. Three independent experiments were performed, the student’s t-test was utilized to determine the p-value and the asterisk (*) indicates a significant change compared with control group. b IHC staining was performed to evaluate BECN1 expression in 20 paired mice lung tissues with 20 Gy radiation and their normal tissues without 20 Gy radiation. The IHC images were captured using the AxioVision Rel.4.6 computerized image system. c An autophagy flux experiment was performed to test the autophagy process. The protein levels of BECN1 and LC3B in MDA-MB-231 cells were detected by western blotting analysis. GAPDH was used as the control. MDA-MB-231 cells cotreated with or without 3-MA were harvested at the indicated timepoints after 4-Gy γ-ray-irradiation. d An autophagy flux experiment was performed to test the autophagy process. The protein levels of BECN1 and LC3B in MDA-MB-231 cells were detected by western blotting analysis. GAPDH was used as the control. MDA-MB-231 cells cotreated with or without 3-MA were harvested at the indicated timepoints after4-Gy γ-ray-irradiation. e The effect of BECN1 knockout on autophagy flux was examined. The cells were transfected with tandem GFP-LC3-mRFP-LC3ΔG plasmids mediated by the adenovirus, and fluorescence laser confocal microscopy was performed(bars: ×20 magnification). f Quantitative measurement of GFP and mRFP. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 compared with the WT cells. g Western blot assay was performed to detect the radiation-induced EMT biomarkers’ alteration post 4 Gy radiation in MDA-MB-231 cells at indicated timepoints. 8 Fig. 3 A-H Complete each panel in Fig 3. with 1) cell lines or models- if not shown in the Fig. , 2) Key points with arrows or boxes , 3) assay used, 4) drug or other treatments if not shown in the fig and 5) abbreviations used spelled out 9 Fig. 3 A-H Panel A. Panel B. Panel C. Panel D. Panel E. Panel F. Panel G. Panel H. Fig. 3: Involvement of autophagy in the regulation of IR-induced cell-cycle progression arrest. a Representative flow cytometry histogram of cell-cycle progression in the population of 4-Gy γ-ray-irradiated HeLa cells with or without cotreatment with 3-MA. b Effect of 3-MA on the G2 and M phase distribution in the population of irradiated cells. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 compared with the control group. c Representative flow cytometry histogram of cell-cycle progression in the population of 4-Gy γ-ray-irradiated BECN1-WT and BECN1-KO MDA-MB-231 cells. d Western blotting analysis of BECN1 in BECN1-WT MDA-MB-231 cells (control) and BECN1-KO cells generated by CRISPR/Cas9. GAPDH served as the internal loading control. e proportions of 4-Gy γ-ray-irradiated BECN1-KO and control cells in the G2 and M phases at the indicated times after IR. The data are presented as the means ±Quantitative measurement of the  SDs from three independent experiments; *p < 0.05 compared with the control group. f Representative flow cytometry histogram of cell-cycle progression in the population of 4-Gy γ-ray-irradiated ATG7-WT and AG7-KO MDA-MB-231 cells. g Western blotting analysis of ATG7 in ATG7-WT MDA-MB-231 cells (control) and ATG7-KO cells generated by CRISPR/Cas9. GAPDH served as the internal loading control. h Quantitative measurement of the proportion of 4-Gy γ-ray-irradiated ATG7-KO and control cells in the G2 and M phases at the indicated times after IR. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 compared with the control group. 10 Fig. 4. A-G Complete each panel in Fig 4. with 1) cell lines or models- if not shown in the Fig. , 2) Key points with arrows or boxes , 3) assay used, 4) drug or other treatments if not shown in the fig and 5) abbreviations used spelled out Panel A. Panel B. Panel C. Panel D. Panel E. Panel F. Panel G. Fig. 4. A-G Fig. 4: Autophagy-related BECN1 deficiency inactivates the G2/M checkpoint in response to IR. a Representative flow cytometry histograms of phosphorylated histone H3 (pHH3)-positive mitotic cells in the population of γ-ray-irradiated HeLa cells with or without 3-MA cotreatment. The cells were immunostained with pHH3(Ser10) antibody to detect the proportion of pHH3-positive mitotic cells at the indicated times after IR. b Quantitative measurement of pHH3(Ser10)-positive cells. The data are presented as the means ± SDs from three independent experiments; *p < 0.01 compared with the control group. c Representative flow cytometry histogram of the population of γ-ray-irradiated BECN1-WTor BECN1-KO MDA-MB-231 cells. The cells were immunostained with pHH3(Ser10) antibody to detect the proportion of pHH3-positive cells at the indicated times after IR. A rescue experiment was conducted by transfecting BECN1-KO MDA-MB-231 cells with lentiviral vectors expressing BECN1. d Quantitative measurement of pHH3(Ser10)-positive cells. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 compared with the WT cells. e Representative image of mitotic protein monoclonal-2 (MPM2)-positive cells obtained through immunofluorescence staining (bars: ×20 magnification). f Quantification of MPM2 antibody-stained mitotic cells. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 compared with the WT cells. g pHH3 was detected by Western blotting analysis. β-actin served as the internal control. 12 Fig. 5 A- F Complete each panel in Fig 5. with 1) cell lines or models- if not shown in the Fig. , 2) Key points with arrows or boxes , 3) assay used, 4) drug or other treatments if not shown in the fig and 5) abbreviations used spelled out Fig. 5 A- F Panel A. Panel B. Panel C. Panel D. Panel E. Panel F. Fig. 5: Autophagy inhibition and BECN1 deficiency disturb the response of G2/M checkpoint-related proteins to IR. a Western blotting analysis of G2/M checkpoint-regulating proteins (pATM, pCHK2, and CHK2) in the γ-ray-irradiated A549 cells that were cotreated or not cotreated with the autophagy inhibitor 3-MA. The proteins were detected at the indicated timepoints after IR, and GAPDH served as the internal control. b Western blotting analysis of the phosphorylation of G2/M checkpoint-regulating proteins (pCDC25C, pCDK1, and CDK1) in the γ-ray-irradiated A549 cells with or without cotreatment with the autophagy inhibitor 3-MA. The phosphorylation analysis was performed at the indicated timepoints after IR, and GAPDH served as the internal control. c Western blotting analysis of G2/M checkpoint-associated phosphorylated proteins (pCHK1, CHK1, pWEE1, and pMYT1) in the γ-ray-irradiated BECN1-WTor BECN1-KO MDA-MB-231 cells. GAPDH served as the internal control. d Western blotting analysis of G2/M checkpoint-associated phosphorylated proteins (pCHK2, CHK2, pCDK1, and CDK1) in the γ-ray-irradiated BECN1-WTor BECN1-KO MDA-MB-231 cells. GAPDH served as the internal control. e Quantitative measurement of the levels of pATM, pCHK2, pCDC25C, and pCDK1 in A549 cells with or without 3-MA cotreatment detected at the indicated time point after IR. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 between different groups. f Quantitative measurement of phosphorylated protein levels of pCHK1, pWEE1, pMYT1, and pCDK1 in BECN1-WT and BECN1-KO MDA-MB-231 cells at the indicated timepoints after IR. The data are presented as the means ± SDs from three independent experiments; *p < 0.05 between different groups. 14 Fig. 6 A-D Complete each panel in Fig 6. with 1) cell lines or models- if not shown in the Fig. , 2) Key points with arrows or boxes , 3) assay used, 4) drug or other treatments if not shown in the fig and 5) abbreviations used spelled out 15 Fig. 6 A-D Panel A. Panel B. Panel C. Panel D. Fig. 6: BECN1 deficiency and autophagy inhibition inhibits the dissociation of CDC25C from the CDK1 complex after irradiation. a The levels of phosphorylated CDK1-pY15 protein in BECN1-KO and BECN1-WT MDA-MB-231 cells were detected 1 and 2 h after 4-Gy irradiation by western blot analysis. β-actin served as the internal loading control. b Effects of 3-MA treatment on the protein–protein interactions of the CDK1 complex in MDA-MB-231 cells after irradiation. Cell lysates were collected from DMSO-treated MDA-MB-231 cells and 3-MA-treated cells at 2 h after 4-Gy irradiation, and immunoprecipitates were prepared with anti-CDK1 or anti-IgG antibodies. Western blot analysis was performed using anti-CDK1, anti-CDC25C, and anti-WEEl antibodies. β-actin was used as the internal loading control. c Effects of BECN1 deficiency on the protein–protein interactions of the CDK1 complex after irradiation. Cell lysates were collected from BECN1-WT and BECN1-KO MDA-MB-231 cells at the indicated timepoints after 4-Gy irradiation, and immunoprecipitates were prepared with anti-CDK1 or anti-IgG antibodies. Western blotting analysis was performed using anti-CDK1, anti-CDC25C, and anti-WEEl antibodies. β-actin was used as the internal loading control. d Effects of BECN1 deficiency and overexpression on the protein–protein interactions of the CDK1 complex in MDA-MB-231 cells with or without 4-Gy irradiation. Cell lysates were collected from BECN1-WT cells, BECN1-KO cells, and BECN1-KO cells transfected with exogenous BECN1 mediated by lentiviral vectors with or without 4-Gy irradiation, and immunoprecipitates were prepared with anti-CDK1 or anti-IgG antibodies. Western blotting analysis was performed using the indicated antibodies. β-actin was used as the internal loading control. 16 Fig. 7 A-F Complete each panel in Fig 7. with 1) cell lines or models- if not shown in the Fig. , 2) Key points with arrows or boxes , 3) assay used, 4) drug or other treatments if not shown in the fig and 5) abbreviations used spelled out 17 Fig. 7 A-F Panel A. Panel B. Panel C. Panel D. Panel E. Panel F. Fig. 7: The radiation-induced cytoplasmic translocation of CDC25C is blocked under BECN1-deficient conditions. a The levels of CDC25C and BECN1 proteins in the cytoplasm and nucleus, respectively, in BECN1-KO and BECN1-WT MDA-MB-231 cells after 4-Gy irradiation were detected by Western blot analysis. β-actin served as the internal loading control. Tubulin and LAMIN A/C proteins were detected as the control cytoplasmic and nuclear proteins, respectively. Exogenous BECN1-expressing vectors were transfected into BECN1-KO cells for the rescue experiment. b CDC25C and CDK1 in BECN1-WT and BECN1-KO MDA-MB-231 cells with or without 4-Gy γ-ray-irradiation were detected by immunofluorescence confocal microscopy(bars: ×20 magnification). c CDC25C and CDK1 in BECN1-WT and BECN1-KO MDA-MB-231 cells with or without 4-Gy γ-ray-irradiation were detected by immunofluorescence confocal microscopy. Exogenous BECN1-expressing vectors were transfected into BECN1-KO cells for the rescue experiment(bars: ×20 magnification). d. The BECN1 levels after 4-Gy γ-ray-irradiation with or without 3-MA treatment were assayed by immunofluorescence microscopy (bars: ×20 magnification). e The levels of BECN1, tubulin and LAMIN A/C proteins in the cytoplasm and nucleusafter 4-Gy irradiation with or without cotreatment with 3-MA were detected by Western blot analysis. β-actin served as the internal loading control. f Effects of siRNA-mediated knockdown of ATG5 and ATG7 on the radiation-induced nuclear translocation of BECN1 (cells???) 18 Fig. 8. A-F Complete each panel in Fig 8. with 1) cell lines or models- if not shown in the Fig. , 2) Key points with arrows or boxes , 3) assay used, 4) drug or other treatments if not shown in the fig and 5) abbreviations used spelled out 19 Fig. 8. A-F Panel A. Panel B. Panel C. Panel D. Panel E. Panel F. Fig. 8: BECN1 mediates the interaction of CDC25C with CHK2. a The interactions of BECN1 with CDC25C, CHK2, CHK1, 13-3-3α, WEE1, CDK1, CCNB1, and BCL2in MDA-MB-231 cells with or without 4-Gy irradiation were assayed, and immunoprecipitates were prepared with anti-BECN1 or anti-IgG antibodies. Western blot analyses were performed using the indicated antibodies. b The interactions of BECN1 with CDC25C and CHK2 in either the cytoplasm or nucleusof MDA-MB-231 cells with or without 4-Gy irradiation were assayed, and immunoprecipitates were prepared with anti-BECN1, anti-CHK2, anti-CDC25C, or anti-IgG antibodies. Western blot analysis was performed using anti-CDC25C and anti-CHK2 antibodies. c BECN1-truncated mutants were constructed according to the listed illustrator. d Flag-BECN1, flag-BECN1-A, flag-BECN1-B, flag-BECN1-C, flag-BECN1-D, flag-BECN1-E, and empty vector were overexpressed in MDA-MB-231 cells. Immunoprecipitation was performed using a flag antibody. e Flag-BECN1, flag-BECN1-A, flag-BECN1-B, flag-BECN1-C, flag-BECN1-D, flag-BECN1-E, and empty vector were overexpressed in BECN1-KO MDA-MB-231 cells. Immunoprecipitation was performed using anti-CDC25 antibody, and western blot analysis was performed using anti-CDC25C and anti-CHK2 antibodies. f The cells (??????) were transfected with the indicated Flag- or HA-tagged expression vectors, and the immunoprecipitations were performed using anti-HA, anti-Flag, or anti-IgG antibodies. Western blot analyses were performed using anti-Flag, anti-HA, anti-CDK1, and anti-CCNB1 antibodies. β-actin served as the internal loading contro 20