Anti tfrc

WB was conducted according to conventional protocols. Briefly, cells treated as indicated were harvested and lysed using RIPA buffer (Beyotime, China). Proteins were extracted and transferred to nitrocellulose membranes via SDS–PAGE. Then, membranes were blocked with 5% skimmed milk for 1 h at room temperature prior to incubation with primary antibodies overnight at 4 °C. The primary antibodies used were anti-O-GlcNAc (Abcam, #ab2735), anti-OGT (Abcam, #ab184198 or #ab177941), anti-YAP (Abcam, #ab52771), anti-β-Tubulin (CST, #2128), anti-Histone-H3 (Santa Cruz, #sc-10809), anti-TFRC (Abcam, #ab84036), anti-SLC7A11 (Abcam, #175186), and anti-GAPDH (CST, #5176). For nuclear and cytosolic separation, nuclear extraction was conducted according to the manufacturer’s protocols for the Nuclear Extraction Kit (Active Motif, USA). Membranes were incubated with HRP-linked secondary antibodies [anti-rabbit (CST, #7074) or anti-mouse (CST, #7076)] for 1 h at room temperature. Signals were detected using Pierce™ ECL Western Blotting Substrate (Thermo Scientific, USA).

The GC cell lines NUGC-3, MKN-1, MKN-45, HGC-27, and NUGC-4 were obtained from Shanghai Bioleaf Biotech Co., Ltd. (Shanghai, China). Cells were cultured in RPMI 1640 medium (Kino Biological and Pharmaceutical Technology Co., Ltd., Hangzhou, China) containing 10% foetal bovine serum (FBS, Gibco, Grand Island, USA) and 1% penicillin/streptomycin (Kino Co., Ltd., Hangzhou, China) in a humidified atmosphere containing 5% CO2/95% air at 37 °C. The antibodies anti-GPx4 (Cat No. ab125066), anti-CDK4 (Cat No. ab108357), anti-CDK4 (Cat No. ab124821), anti-Cyclin D1 (Cat No. ab16663), anti-Ferritin Heavy chain (Cat No. ab287968) and anti-TFRC (Cat No. ab214039) were purchased from Abcam (Cambridge, UK). Anti-LC3B (Cat# 2775) antibody was purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-NCOA4 (Cat. Sc-373739) was purchased from Santa Cruz Biotechnology (Texas, USA).