Ab229914

Briefly, after performing the indicated treatments, all proteins were extracted with RIPA buffer (Solarbio, Beijing, China), and protein quantification was performed with a BCA assay kit (Solarbio, Beijing, China). Protein lysates were separated by SDS–polyacrylamide gel electrophoresis (PAGE) electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). PVDF membranes were incubated overnight at 4°C with rabbit anti‐ARID1A (1:1000, 12354S, CST), rabbit anti‐CDK2 (1:10,000, ab32147, Abcam), rabbit anti‐CDK4 (1:1000, DF6102, Affinity), rabbit anti‐CDK6 (1:100,000, ab124821, Abcam), rabbit anti‐Cyclin D (1:2000, AF0931, Affinity), mouse anti‐GAPDH (1:50,000, 60004‐1‐Ig, Proteintech), rabbit anti‐BAX (1:1000, 41162S, CST), rabbit anti‐Cleaved Caspase‐3 (1:1000, 9664S, CST), rabbit anti‐Cleaved Caspase‐7 (1:1000, 8438S, CST), rabbit anti‐β‐actin (1:10,000, AF7018, Affinity), rabbit anti‐RAD50 (1:5000, 29390‐1‐AP, Proteintech), rabbit anti‐CHK2 (1:1000, 6334S, CST), rabbit anti‐RAD51 (1:2000, 14961‐1‐AP, Proteintech), rabbit anti‐γ‐H2AX (1:1000, ab229914, Abcam), rabbit anti‐BRG1 (1:1000, 49360S, CST), followed by a 1 h incubation at room temperature with the horseradish peroxidase (HRP)‐conjugated respective secondary antibody for chemiluminescence‐based protein detection.

HepG2 and Hep3B cells were plated onto glass coverslips coated with gelatin in 24-well plates. Then, cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.25% Triton-X 100 for 15 min, and blocked with 5% BSA in PBS for 30 min. Subsequently, cells were incubated with anti-γH2AX antibody (1:1000 dilution, ab229914, abcam, USA) for 1.5 h, followed by incubation with Alexa Fluor® 647 conjugated goat anti-rabbit IgG (1:1000 dilution, ab150079, abcam, USA) for 1 h. Nuclear was counterstained with 1 µg/mL DAPI solution (Thermo Fisher Scientific, USA). Stained cells were visualized with a Zeiss LSM700 confocal microscope (Carl Zeiss, Germany).

Antibodies against glutathione-S-transferase (GST, 10000-0-AP), poly (ADP-ribose) polymerase 1 (PARP1, 13371-1-AP), Flag (20543-1-AP), MYC-tag (16286-1-AP), GFP (66002-1-Ig), Ki-67 (27309-1-AP), histone-H3 (17168-1-AP), topoisomerase 1 (TOP1, 20705-1-AP), and caspase 3 (19677-1-AP) were purchased from Proteintech (Wuhan, China); PAR (AM80), β-actin (A5441), and methyl methanesulfonate (MMS, M4016) were obtained from Sigma–Aldrich (St. Louis, MO, USA); mouse IgG (A7028) and rabbit IgG (A7016) were purchased from Beyotime (Shanghai, China). Antibodies for γH2AX (ab22551), H2AX (ab229914), HMGB3 (ab75782), and Rad51 (ab63801) were obtained from Abcam (Cambridge, UK). Goat anti-rabbit IgG Alexa Fluor-488 (A-11008) and Goat anti-mouse IgG Alexa Fluor-594 (A-11030) for immunofluorescence staining were obtained from Invitrogen (Waltham, MA, USA). Olaparib (AZD2281) was purchased from Selleck Chemicals (Houston, TX, USA).

IHC staining was employed to investigate the expression of APOBEC3A protein in formalin-fixed, paraffin-embedded tissue sections as described previously. Primary antibody against APOBEC3A (#AP20219a, abcepta) was used overnight at 4°C. Slides were then incubated for another 1 h using secondary antibody (#PK-8501, Vector Lab, USA). The complex was detected using Rabbit IgG mini-PLUS Kit visualized with DAB complex (#PK-8501, Vector Lab, USA). Besides, hematoxylin was applied to counterstain the nuclei. Sections were visualized under a microscope (10× or 40×). For immunofluorescence, the transfected cells were plated onto coverslips for 24 h and fixed for 20 min with 4% paraformaldehyde. After permeabilization for 30 min with 0.5% Triton X-100, cells were incubated in blocking buffer. Primary antibody against H2Ax (1:500, #ab229914, abcam, US) was utilized overnight at 4°C, then washed three times with PBS for 3 min. Alexa-488 goat anti-rabbit (1:500, #GB25303, Servicebio, China) was used as secondary antibody for 60 min and stained with 4’,6-diamidino-2-phenylindole (DAPI) for 15 min at room temperature. Finally, cells were regarded as positive if at least one focus was visible using a 40× objective in a confocal microscope.

Deoxypodophyllotoxin (DPT) was obtained from Yuanye Bio-technology company (Shanghai, China). It was dissolved in DMSO and then stored at the concentration of 10 mmol/L. MnTBAP was from Sigma (St. Louis, MO, USA) and 3-Aminobenzamide (3AB), NAD⁺ and FK866 were all from MedChemExpress Company (Shanghai, China). The antibodies against TAX1BP1 (bs-13671R), NDUFS2 (bs-10455R) and NDUFS4 (bs-3961R) were all obtained from Bioss Antibodies (Beijing, China), against PARP1 (13371-1-AP), ND2 (19704-1-AP) and ND1 (19703-1-AP) were all from Proteintech Company (LA, USA), and against AIF (ab32516), GPX4 (ab125066), catalase (ab76024), H2AX (ab229914), TOMM20 (ab186735), phospho-H2AX at Ser139 (ab26350) and phospho-ATM at Ser1981 (ab81292) were all from Abcam company (Cambridge, UK), against PAR (#83732) and β-Actin (#4970) were both from Cell Signaling Technology Company (Beverly, MA, USA). Second antibody against rabbit (A0208) and against mouse (A0216) were from Beyotime Biotechnology (Nanjing, China).

Brain sections were cut from paraffin‐embedded brain tissue blocks. Paraffin‐embedded tissue sections were used for H&E staining. For IHC, brain sections were dewaxed, hydrated, treated in 1 × EDTA buffer (Zsbio) at 100°C for 20 min for antigen retrieval, then incubated with goat serum at room temperature for 30 min, followed by overnight incubation at 4°C with rabbit anti‐ARID1A (1:2000, 12354S, CST), rabbit anti‐Ki67 (ZM‐0167, ZSGB‐BIO), rabbit anti‐p‐ERK1\2 (1:300, 4370S, CST), rabbit anti‐RAD51 (1:500, 14961‐1‐AP, Proteintech), rabbit anti‐γ‐H2AX (1:200, ab229914, Abcam), The next day, slides were rinsed with 1 × PBS three times, followed by incubation with enzyme‐labeled secondary antibody at room temperature for 1 h. Color development was performed using diaminobenzidine (DAB) reagent, followed by image acquisition under brightfield microscopy.