Ab81299

Direct immunofluorescence staining was employed to assay the DNA damage of HGEC induced by extracts. Briefly, HGEC were replated into 24-well plates at 5 × 10⁴ cells/well for 1 day and then exposed to extracts for 24 h at 37°C. Cells were rinsed three times with PBS, fixed by 4% poly-formaldehyde for 30 min, and permeabilized by 10% Triton X-100 for 15 min. After being washed three times with PBS, cells were blocked with 1% bovine serum albumin (BSA) for 60 min. Subsequently, cells were incubated overnight at 4°C with the rabbit monoclonal antibody anti-γ-H2AX (ab81299, Abcam, USA). After being maintained at room temperature for 1 h, cells were immersed in PBS for 5 min, then incubated by Goat Anti-Rabbit IgG (H+L) Fluor488-conjugated (Affinity Biosciences, USA) at room temperature for another 1 h. Last, the cells had a 10 min dark incubation at 37°C with 4', 6-diamidino-2-phenylindole (DAPI, Service bio, China) for nuclear counterstaining. Immunofluorescent images were observed and photographed with an inverted microscope system (IX73, Olympus, Japan).

AML12 cells were fixed with fresh 4% paraformaldehyde for 10 min, and were permeabilized with 0.1% Triton X-100 in PBS for 10 min. After blocking with 1% BSA and 22.52 mg/mL glycine in PBS for 1 h, anti-Phospho-ATM (Ser1981) (1:50, sc-47739, Santa Cruz BioTech, Santa. Cruz, CA, USA) and anti-γH2AX (1:100, ab81299, Abcam, Cambridge, UK) were added and incubated overnight at 4 °C. The following day, plates were washed three times with PBS. After rinsing, secondary antibodies were applied for 1 h at room temperature, followed by three additional washes with PBS. Nuclei were counterstained with DAPI. Images were acquired by the EVOS M7000 Image System with a 40× objective.

Cells were fixed with 4% paraformaldehyde/PBS for 15 min and permeabilized with 0.5% Triton X-100 (in 1 × PBS) for 20 min at room temperature. Primary antibodies for staining CTPS2 (ab32087, Abcam), BRCA1 (Santa Cruz, TX, USA) and p-H2AX (ab81299, Abcam) were used at 1:200 for 1 h at room temperature. After three washes with PBS, the sections were incubated with the secondary antibody in PBS with 2% normal serum for 1 h at room temperature, followed by three washes in PBST. Subsequently, cells were stained with 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI; a DNA-specific fluorescent dye) for 5–10 min. Stained cells were covered with coverslips and visualized using confocal microscope.

Liver tissue proteins were obtained from tissue lysates for western blotting. Protein concentration was determined using a BCA assay kit (Sigma-Aldrich, Saint Louis, MO, USA). Denatured proteins were separated on 12–20% Bis-Tris MOPS gels by SDS-PAGE and transferred to PVDF membranes. The PVDF membranes were incubated overnight at 4 °C with anti-Phospho-SAPK/JNK (Thr183/Tyr185) (1:1000, #4668, CST, Boston, MA, USA), anti-JNK (1:1000, #9252, CST, Boston, MA, USA), anti-VDAC1 (1:2000, ab154856, Abcam, Cambridge, UK), anti-Phospho-Src Family (Tyr416) (1:1000, #6943, CST, Boston, MA, USA), anti-Src (1:1000, #2123, CST, Boston, MA, USA), anti-Phospho-ATM (Ser1981) (1:400, sc-47739, Santa Cruz BioTech, Santa. Cruz, CA, USA), anti-ATM (1:4000, A1106, Sigma-Aldrich, MO, USA), anti-γH2AX (1:5000, ab81299, Abcam, Cambridge, UK), anti-H2AX (1:1000, 10856-1-AP, Proteintech, Wuhan, China), anti-p21(1:1000, ab188224, Abcam, Cambridge, UK) and anti-GAPDH (1:10,000, MB001, Bioworld Technology, Nanjing, China). The membrane-bound antibodies were detected by a hypersensitive chemiluminescence detection reagent from Fude Biological Technology Co., Ltd. (Hangzhou, China).

Antibodies against CDK1 (ab133327), Cyclin B1 (ab32053) and CDC25C (ab32444), Caspase-3 (ab32351), Bcl2 (ab182858), LC3B (ab192890), SQSTM1/P62 (ab109012), and γH2AX (ab81299) were obtained from Abcam (Cambridge, MA, USA). Antibodies against NEK2 (66632-1-lg), TRIM21(12108-1-AP), α-tubulin (11224-1-AP), GAPDH (60004-1-Ig), Bax (60267-1-Ig), Alexa Fluor 594-conjugated donkey anti-rabbit second antibody (SA00013-4), and Alexa Fluor 488-conjugated donkey anti-mouse second antibody (SA00013-1) were obtained from Proteintech (Wuhan, China). An antibody against NEK2 (sc55601) was got from Santa Cruz Biotechnology, Inc (St Louis, MO, USA). The autophagy activator Rapamycin (RAPA, HY-10,219), Cycloheximide (CHX, HY-12,320), and MG132(HY-13,259) were purchased from MedChemExpress (MCE, USA).

Immunoprecipitation was conducted according to the instructions provided in a Co-IP Kit (Roche). The L3/4/5 dorsal root ganglions (DRGs) were isolated from sham and SNI with mitochrondrial injection therapy (SNI + Mito) groups. Each Co-IP sample consisted of 18 pooled DRGs from 6 mice per group. The protein extracts from DRGs were prepared following the same procedure as described for Western blot analysis. To eliminate any non-specific binding, the samples were incubated with protein G-agarose on a rotator at 4 °C for 3 h. After centrifugation, the supernatant was transferred to fresh tubes and mixed with 5 µl of anti-γH2AX antibody (#ab81299, Abcam) for 1 h. Subsequently, a homogeneous suspension of protein G-agarose (50 µL) was added and left to incubate overnight at 4 °C on a rotator. Following centrifugation and removal of the supernatant, the beads were washed twice with lysis buffer 1, twice with lysis buffer 2, and once with lysis buffer 3. Protein sample buffer was then added before boiling the samples for 5 min. The presence of CTCF in immunoprecipitated proteins was detected using an anti-CTCF antibody (#ab188408, Abcam) through Western blot analysis.

Immunofluorescence staining for expression of γ‐H2AX was conducted as described in a previous study (Xu, Xu, et al., 2019 (link)). Briefly, sections cut from the testes in different groups were incubated with 5% fetal bovine serum for 30 min at room temperature and then incubated overnight at 4°C with primary antibodies against γ‐H2AX (ab81299, Abcam, 1:200). Goat anti‐mouse Alexa Fluor 488 (A0428, Beyotime, CHN) was used for secondary staining. Nuclei were counterstained with DAPI (G1012, Servicebio, CHN). Images of the stained samples were captured using a fluorescence microscope (Olympus, Tokyo, Japan). The γH2AX expression was analyzed from the images of IF staining by using ImageJ software 1.8.0 (National Institutes of Health, United States). The data were expressed as a percentage of the fluorescence of the control group.