P0018am

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).

The lower lobe and posterior lobe of lung were preserved at −80°C and analyzed by western blotting for molecular biology. Lung tissues were ground thoroughly using RIPA lysis buffer (Solarbio, Beijing, China) under an ultrasonic crusher to extract the proteins, and added loading buffer. The proteins were separated using gel electrophoresis and moved onto membranes, which was sealed with 5% skim milk for 1 h and specific primary antibodies (Proteintech, Wuhan) were added to soak membranes at 4°C for overnight, including GAPDH (1:10000), GSK-3β (1:5000), cyclin D1 (1:8000), β-catenin (1:10000), Bax (1:8000), and Bcl2 (1:1500). Then, using secondary antibody (IgG, 1:5000, Proteintech, Wuhan) to incubate the membrane and observe the imprinting via an ultrasensitive ECL chemiluminescence kit (Beyotime, P0018AM, Shanghai, China). Finally, blots were quantified by ImageJ software.