Ncoa4

HepG2 cell line was obtained from HonorGene (Changsha, China). Ferrostatin-1, MTT, 3-methyladenin (3-MA), chloroquine (CQ), RPMI-1640, and other chemicals were purchased from Sigma-Aldrich. 2-Pyridylhydrazone dithiocarbamate s-acetic acid (PdtaA) was prepared in our laboratory [27 (link)]. GPx4, xCT (SLC7A11), NDRG1, vimentin, and NCOA4 antibody were obtained from the Proteintech Group (Wuhan, China). Antibodies of E-cadherin and secondary antibodies (fluorescence-labeled for immunofluorescence) were purchased from Cell Signaling Technology (USA). Ferritin antibody for immunofluorescence was obtained from Santa Cruz Biotechnology (USA, Santa Cruz). NCOA4 antibody for immunofluorescence was purchased from Atlas Antibody (Sweden). Secondary antibodies for Western blotting were obtained from EarthOx, LLC (San Francisco, USA).

3-Methyladenine (3-MA), dichlorofluorescein (H₂DCF-DA), ferrostatin-1, 4′,6-diamidino-2-phenylindole (DAPI), TGF-β1, N-acetyl-L-cysteine (NAC), Roswell Park Memorial Institute- (RPMI-) 1640, and other chemicals were purchased from Sigma-Aldrich (USA). Erastin was from MedChemExpress. Antibodies of vimentin, NCOA4, Nrf2, Keap1, HO-1, and gapdh for Western blotting were obtained from Proteintech Group Inc. (Wuhan, China). Antibodies of E-cadherin and ferritin (H chain) and secondary antibodies (fluorescence labeled for immunofluorescence) were purchased from Cell Signaling Technology (USA). Ferritin antibody for immunofluorescence was obtained from Santa Cruz Biotechnology (USA, Santa Cruz). NCOA4 antibody for immunofluorescence was purchased from Atlas Antibody (Sweden). Secondary antibodies for Western blotting were obtained from EarthOx, LLC (San Francisco, USA).

4T1 cells were pre-seeded in a 6-well culture plate. Cells were washed twice with PBS solution and then collected using cell lysate, PMSF, and protease inhibitor. The contents of protein were measured using the BCA protein concentration assay kit. According to the required sample volume, 5 × loading buffer was added into the protein solution at a ratio of 4: 1. Then the final protein solution was denatured at 100 ℃ for 10 min in a boiling water bath. The separation gel and concentrated gel were prepared, and the samples were loaded at 30 µg of protein volume, electrophoretically separated and transferred into the polyvinylidene fluoride (PVDF) membrane, and then closed by 5% skim milk solution at room temperature for 2 h. These samples were incubated with the corresponding primary antibody such as β-actin (1: 20,000, Proteintech), SLC7A11 (1: 1000, Origo), GPX4 (1: 1000, Proteintech), 4- HNE (1: 1000, Origo), LC3 (1: 1000, Proteintech), NCOA4 (1: 1000, Proteintech), FTH1 (1: 1000, Proteintech), and γ-H2AX (1: 1000, Proteintech) overnight at 4 °C. After that, these samples were cultivated with the secondary antibody (1: 10,000, Proteintech) for 2 h. At last, the membranes were visualized and processed with image J for grayscale values.