Fig 2: Knockdown of MCM2 increases the protein expression levels of γ-H2AX and p53 after treatment with carboplatin. (A) A2780 cells were treated with various concentrations of carboplatin (0, 20, 30 and 40 µg/ml) for 48 h. Subsequently, the cells were lysed and the total protein was extracted. Protein expression levels of MCM2, p53, γ-H2AX and total H2AX were detected by western blotting. MCM2 knockdown increased DNA damage-associated markers in response to carboplatin. (B) Quantification of the protein expression levels of MCM2, (C) p53 and (D) the γ-H2AX/H2AX ratio. γ-H2AX was used as an indicator of double-strand breaks. Data are presented as the mean ± standard deviation from three experiments. *P<0.05 and **P<0.01 vs. the respective control. MCM2, minichromosome maintenance complex component 2; shRNA, short hairpin RNA; shCON, control shRNA; shMCM2, shRNA targeting MCM2; H2AX, H2A histone family member X.

Fig 3: LINC00261 overexpression enhances EC radiosensitivity in vivo. TE-1-R cells with stable oe-LINC00261 were injected into mice. Tumor volume (A) and weight (B) were measured. (C and D) miR-552-3p (C), and DIRAS1 mRNA (D) expressions in mice tumor tissues were detected by RT-qPCR. (E) ?-H2AX and Ki67 expressions in tumors were measured by IHC. (A and B) N = 12; (C and D) N = 6. The measurement data were presented as mean ± standard deviation. One-way ANOVA was appointed to analyze data in (B–D). Two-way ANOVA was appointed to analyze data in (A and E). Tukey’s multiple comparisons test was employed for the post hoc test. *p < 0.05.

Fig 4: Identification of etoposide-induced ARP8 phosphorylation and the possible responsible kinase.(A) Amino acid sequence 408 through 420 of ARP8. The Ser412 residue, within the ATM/ATR substrate motif and the CK2 substrate motif, is indicated. (B) Immunoprecipitation analysis of ARP8 phosphorylation. U2OS cells transiently expressing an empty HA vector or a vector encoding HA-tagged ARP8 were treated with DMSO (ctrl) or etoposide (Etp) for 15 min, then washed twice and cultured in complete medium for the indicated times. The nuclear extracts were incubated with anti-HA-conjugated anti-mouse IgG Dynabeads. The precipitates were electrophoresed through a gel and probed by western blotting with an anti-ATM/ATR substrate antibody or an anti-HA antibody. λPPase treatment identified the band of phosphorylated HA-ARP8. The blot of input was probed by antibodies against phospho-ATM (p-ATM), γ H2AX or phospho-RPA2 at Ser4/8 (p-RPA2). β-actin was used as a loading control. (C) Identification of the ARP8 phosphorylation site by an immunoprecipitation analysis. U2OS cells were transfected with an empty HA vector (vet), or a vector encoding HA-tagged wild-type ARP8 (WT) or HA-ARP8 S412A (S412A) for 48 hr. The cells were washed after treatment with etoposide or DMSO for 15 min, cultured in fresh medium, and harvested at the indicated time points. Whole cell extracts were used for the immunoprecipitation analysis. (D) Etoposide-induced ARP8 phosphorylation in ATM-deficient BIVA and ATM-proficient 11–4 cells. Immunoprecipitation analysis of cell extracts of BIVA or 11–4 cells transfected with HA-tagged wild-type ARP8 using anti-HA antibodies. The cells were treated with DMSO (ctrl) or etoposide (Etp) for 15 min, cultured in fresh medium, and harvested at the indicated time points. Whole cell extracts were used for the immunoprecipitation analysis, which was performed as described in (B). The amounts of phosphorylated ARP8 and HA-ARP8 were quantified, using the Image J software. The results of the quantitative analysis are shown as the relative values to the DMSO controls. Source data are presented in Figure 1—source data 1. (E) Immunoprecipitation analysis of cell extracts from 11 to 4 cells expressing HA-tagged ARP8. The cells were treated with DMSO, 10 μM ATMi (KU55933), or 10 μM ATRi (VE821) for 2 hr before etoposide treatment, and then the inhibitors (5 μM) were added after the cells were washed.10.7554/eLife.32222.005Figure 1—source data 1.Source raw data for Figure 1D.

Fig 5: Effect of palbociclib on γ-H2AX and 53BP1 foci formation in irradiated CNE-1 and CNE-2 cells. Cells were seeded on coverslips in a 6-well plate, then treated with radiation and the absence or presence of palbociclib. The palbociclib was administrated 18 hrs before irradiation (palbociclib->RT), at the time of irradiation (RT+ palbociclib), or 6 hrs after irradiation (RT-> palbociclib). γ-H2AX and 53BP1 were detected by immunofluorescence assay for fluorescent foci. Foci were counted in 50 cells per treatment condition. (A) Representative images of nuclei from each group. Scale bars: 10 µm. (B) Histogram represented Mean and SD values for γ-H2AX and 53BP1 foci/nucleus from three independent. ***P <0.001.