γh2a x

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.

Indicated cells with different dose of osalmid were lysed in the radioimmunoprecipitation assay (RIPA) buffer, and the detailed procedure was conducted according to the previous report (Yao et al., 2017 (link)). The following antibodies were used: β-actin and γH2Ax (Proteintech, Wuhan, China).