Acsl4

Western blotting and the analysis of the blot results was conducted as previously reported^{27 (link)}. In brief, cells were lysed and centrifuged at 12,000 g for 10 minutes at 4 °C. The supernatants were denatured at 100 °C for 5 minutes and mixed with a 1/5 volume (corresponding to the volume of the supernatants) of loading buffer (Beyotime, China). Then, 30-µg protein samples were loaded into each well in a 10% SDS-polyacrylamide gel and were subjected to electrophoresis. Once the proteins of diverse molecular weights were sufficiently separated, the proteins were transferred to PVDF membranes (Millipore, USA). Next, the PVDF membranes with the transferred proteins were blocked with 5% nonfat milk diluted in TBST for 2 hours at 25 °C and were then incubated with primary antibodies, such as SLC7A11, SLC3A2, GPX4, GSS, GCLC, ACSL4 (Cell Signaling Technology, USA) and β-actin (Beyotime, Shanghai, China), with dilutions of 1:1500, 1:2000, 1:2000, 1:2500, 1:3000 and 1:10,000, respectively, at 4 °C for 12 hours. Next, the membrane was washed again with TBST and was incubated with a 1:10,000 dilution of horseradish peroxidase (HRP)-labelled anti-rabbit IgG (Beyotime, Shanghai, China) for 1 hour at 25 °C. Finally, the respective protein bands were visualized using enhanced chemiluminescence reagents (BOSTER, Wuhan, China).

The whole cell lysates were prepared following established protocols. In summary, cells were lysed using M-PER Mammalian Protein Extraction Reagent (Thermo Scientific Technologies, USA) supplemented with a protease inhibitor cocktail (Thermo Scientific Technologies, USA). The resulting lysates contained total protein. The protein concentration in the lysates was measured using a Bradford assay kit (Beyotime Biotechnology, Shanghai). Antibodies for CPT1A, c-Myc, SOX2, OCT4, NANOG, GPX4, ACSL4, NRF2, Ubiqutin, FBXW7, GAPDH and β-actin (Cell Signaling Technology; 1:1000), were used for western bolt.