Lf102

Protein extracts were prepared with RIPA Lysis and Extraction Buffer (89,901, Thermo Scientific, USA) supplemented with Protease and Phosphatase Inhibitor Cocktail (78,443, Thermo Scientific, USA) according to the manual. Then proteins were separated on SDS-PAGE gels (10%) followed by transfer to polyvinyl difluoride (PVDF) membranes (pore size 0.2 µm, 88,520, Thermo Scientific, USA). The membrane was subsequently blotted in 1% bovine serum albumin (BSA, Sigma-Aldrich St. Louis, MO, USA) in PBS for 2 h and incubated overnight with commercially available primary antibodies against β-actin (1:1000 dilution, 4970S, Cell Signaling Technology, Danvers, MA, USA), Interleukin-6 (IL-6) (1:1000 dilution, 4970S, Cell Signaling Technology, Danvers, MA, USA) and TNF-a (1:2000 dilution, 41,504, Signalway Antibody, Pearland, TX, USA) at 4 °C. After washing three times with PBS containing 0.05% Tween-20, membranes were incubated with secondary antibodies coupled with HRP (1:4000 dilution, LF102, EpiZyme, Shanghai, China) followed by washing three times. The images were captured with Bio-Rad gel imaging system and analyzed by Quantity One software.

The liver tissue was homogenized in lysis buffer. After centrifugation, the protein concentration of the lysate was measured using BCA Protein Assay Kit (Beyotime, P0012, China), in line with the manufacturer's protocols. Equivalent amounts (50 μg) of protein were separated on SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were blocked with 5% skimmed milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and incubated with antibodies against COX2 (1 : 1000, 12375-1-AP, Proteintech), ACSL4 (1 : 1000, A16848, ABclonal), GPX4 (1 : 1000, BM5231, Boster), FTH1 (1 : 1000, A19544, ABclonal), Nrf2 (1 : 1000, 16396-1-AP, Proteintech), HO-1 (1 : 1000, 10701-1-AP, Proteintech), or GAPDH (1 : 10 000, 60004-1-AP, Proteintech) overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies against rabbit or mouse IgG (1 : 5000, LF101 and LF102, respectively, Epizyme) for 1 hour at room temperature. A Bio-Rad immunoblot analysis detection system (Bio-Rad, Hercules, CA, USA) was used for visualization. Protein band densities were assessed by ImageJ analysis software, and the relative densities against the loading control (GAPDH) were calculated.

Cells were washed twice with PBS and subsequently lysed with RIPA buffer containing protease and phosphatase inhibitors (P0013B and P1045, Beyotime, China) on ice for 30 min. Proteins in the lysates were separated by SDS-PAGE and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were then blocked with 5% non-fat milk at room temperature for 1 h and incubated with primary antibodies, including anti-TFRC (66180-1-Ig, Proteintech, China), anti-FTH1 (49644, Signalway Antibody, USA), anti-FTL (10727-1-AP, Proteintech, China), anti-POLQ (Fab4305, Fenghui Antibody, China), anti-RAD51 (67024-1-lg, Proteintech, China), anti-phospho-Histone H2AX(Ser139) (AF5836, Beyotime, China), anti-α-tubulin (66031-1-Ig, Proteintech, China), and anti-β-actin (66009-1-Ig, Proteintech, China), at 4 °C overnight. Unbound antibodies were removed by washing the membranes three times with TBST. Goat anti-rabbit (LF102, Epizyme, China) or anti-mouse (7076 S, Cell Signaling Technology, USA) secondary antibodies were applied at a 1:5000 dilution and incubated at room temperature for 1 h. After another three washes with TBST to remove unbound antibodies, the HRP signaling was detected using an ECL substrate (P0018AM, Beyotime, China) in an illuminance imaging system (Tanon5200, Tanon, China).

For Western blot analysis, total protein was extracted from cultured cells in protein lysis buffer (50 mM Tris–HCl, pH 8.0; 10 mM NaCl; 10% NP-40) containing protease and phosphatase inhibitors (P1045, Beyotime Institute of Biotechnology) on ice. Equal amounts of protein (40 μg/lane) were separated by (10%) SDS–PAGE (120 V) and transferred to polyvinylidene fluoride membranes (FFP24, Beyotime Institute of Biotechnology; 100 V for 120 min), and then incubated first with antibodies against Hdac4 (mouse monoclonal; Cell Signaling Technology, #7628, 1:2,000), Pcna (mouse monoclonal; Cell Signaling Technology, #2586, 1: 2,000), and β-actin (rabbit monoclonal; Cell Signaling Technology, #4970, 1:1,000) and then with HRP-conjugated goat anti-rabbit secondary antibody (LF102, Epizyme, China) and HRP-conjugated goat anti-mouse secondary antibody (LF101, Epizyme). The membranes were washed and bound antibodies were visualized using a chemiluminescence reagent (P0018A, Beyotime Institute of Biotechnology).

Cell cultures and Western blots were performed as described previously [40 (link), 41 (link)]. Human embryonic kidney (HEK) 293T cells obtained from CCTCC were recently tested for mycoplasma contamination but not authenticated. Briefly, HEK293T cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (Hyclone). Cells of about 70%-80% confluence were transfected using lipofectamine 8000 (Beyotime) according to the manufacturer’s instructions. If shRNA was used, the ratio of shRNA to expression constructs is 1:1. Forty-eight hours later, cells were lysed in ice-cold radioimmunoprecipitation (RIPA) buffer containing protease inhibitor cocktail (Thermo Scientific). Samples were then loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to membrane, the samples were then probed with rabbit anti-FLAG antibody (1:1000; #14793, CST) or rabbit anti-GAPDH antibody (1:1000; LF206, Epizyme), developed with species-specific horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000; LF102, Epizyme) and visualized with enhanced chemiluminescence (Epizyme). For all Western blots, three cultures for each group were used.