γh2ax

Cells (1 × 10⁴/well) were fixed with 3.7% formaldehyde (Sigma−Aldrich) for 15 min and permeabilized with 0.2% Triton X-100 for 15 min. Subsequently, cells were blocked with 5% fetal bovine serum in phosphate-buffered saline (PBS; AMRESCO, Solon, OH, USA) for 1 h and then incubated with primary antibodies (1:1000 dilution, 1 h) targeting γ-H2AX, PARP1, p-p53^ser15, and p-ATM (Santa Cruz Biotechnology, Inc.). Cells were then incubated with secondary antibodies (1:1000 dilution, 1 h) conjugated with Alexa Fluor 488 or 546 (Invitrogen, Carlsbad, CA, USA). Nuclei were stained with Hoechst 33258 reagent (5 μM; Sigma−Aldrich) for 15 min. Stained cells were imaged using confocal microscopy (LSM-700, Carl Zeiss Microimaging, Oberkochen, Germany) and analyzed using Zen 2009 software.

The retinas were dissected in PBS and suspended in 120 μL of RIPA buffer (per retina) containing proteinase inhibitor cocktail (Bimake, Shanghai, China, #B14002), protein phosphatase inhibitor A (Beyotime, Shanghai, China, #P1082) and protein phosphatase inhibitor C (Beyotime, Shanghai, China, #P1092). The total proteins were extracted sonication using an EpiSonic 2000 Sonication System (EPIGENTEK, Farmingdale, NY, USA) (Amplitude: 40%, 10 s on and 10 s off for 7 min in total). For Western blot (WB) analysis, it was performed as described previously with some modifications [14 (link)]. For each WB, 30–50 μg of total protein was used. The protein was separated by 12% SDS-PAGE and transferred to the PVDF membrane. The membrane was blocked by 5% milk in TBST for 1 h. After washing with TBST, the membrane was incubated with primary antibodies γH2Ax (Santa Cruz, Dallas, Texas, USA, sc-517348, 1:1000 dilution) and GAPDH (Proteintech, Rosemont, IL, USA, #60004-1-Ig, 1:2000 dilution). The secondary antibody was diluted in TBST (1:3000 dilution). After washing with TBST, enhanced chemiluminescence (ECL) detection was performed by using the Ultra sensitive ECL Chemiluminescence Kit (NCM Biotech, Suzhou, China, #P10300) according to the manufacturer’s specifications. The exposure and development of PVDF membrane were performed using Tanon 5200 (Tanon, Shanghai, China).

GFP (Abcam catalog no. ab6556), TRF1(Abcam catalog no. ab10579), GAPDH (Santa Cruz catalog no. sc-47724), OGG1 (Abcam catalog no. ab124741), Actin Cell Signaling catalog no. 3700), Lamin B1 Abcam catalog no. ab16048), Lamin A/C (Cell Signaling catalog no. 4777), γH2AX (Santa Cruz catalog no. sc-517348), 53BP1 (Novous catalog no. NB100-304), TRF2 (Novous catalog no. NB110-57130), MDM2 (Cell Signaling catalog no. 86934), p53(Santa Cruz catalog no. sc-126), p21 (Cell Signaling catalog no. 2947), p16 (Proteintech catalog no. 10883-1-AP), pRB S807/811 (Cell Signaling catalog no. 8516), pCHK2 T68(Cell Signaling catalog no. 2197), pCHK1 S317 (Cell Signaling catalog no. 12302), pATM S 1981 (Abcam catalog no. ab81292), CHK1 (Cell Signaling catalog no. 2360), H3K27me3 (Cell Signaling catalog no. 9733), H3K27Ac (Cell Signaling catalog no. 8173), LSD1 (Cell Signaling catalog no. 2184), cGAS (Cell Signaling catalog no. 66546), p62 (Cell Signaling catalog no. 39749).

Immunocytochemistry was performed as described previously^{51 (link)}, using antibodies against γH2AX (Santa cruz), 8-OHdG (Nikken Seil), and β-actin (Proteintech). The immunostained samples were observed with either Zeiss LSM800 or Olympus Fluoview FV1000 confocal fluorescence microscope. 8-OHdG intensity was measured in nuclear (DAPI positive areas) and non-nuclear (β-actin positive areas outside of DAPI positive areas) for 20 cells per sample using Image J, and data are shown as mean ± SEM.

Cells were lysed in RIPA lysis buffer (25 mmol/L Tris HCl (pH 7.5), 2.5 mmol/L EDTA, 2.5 mmol/L EGTA, 20 mmol/L NaF, 1 mmol/L Na₃VO₄, 100 mmol/L NaCl, 20 mmol/L sodium ‐glycerophosphate, 10 mmol/L sodium pyrophosphate and 0.5% triton X‐100) supplemented with a protease inhibitor cocktail (Roche). Cellular proteins (30 μg) were separated by SDS‐PAGE and electro‐transferred onto PVDF membranes. Membranes were blocked in Tris‐buffered saline (TBS) containing 5% milk and 0.1% Tween‐20 at room temperature. Membrane incubation with 1° Abs (AhR, CYP1A1, PCNA, γH2AX, PARP and beta‐actin, sourced from Santa Cruz or Cell Signaling) was conducted overnight at 4°C. Membranes were washed at room temperature before incubation with 2° Ab (GE) conjugated with horseradish peroxidase for 1 hour. Detection was performed with Super Signal Chemiluminescent reagent according to the manufacturer's protocol (Tanon, China).