Western Blot Analysis of Protein Expression

As described previously,^{29 (link)} cells were lysed by a lysis buffer. SDS-PAGE (12%, w/v) was used to separate the proteins, which were then transferred onto PVDF membranes (provided by Thermo Fisher, Waltham, MA, USA). Primary antibodies (monoclonal) against GPX4 (cat. no. ab125066, dilution 1:1000, Abcam, Cambridge, UK), p53 (cat. no. ab131442, dilution 1:1000, Abcam), FTH1 (cat. no. ab240277, dilution 1:1000, Abcam), ACSL4 (cat. no. ab227256, dilution 1:1000, Abcam UK) and β- actin (cat. no. ab179467, dilution 1:2000, Abcam), along with peroxidase- conjugated rabbit anti-IgG secondary antibodies (cat. no. A2074, dilution 1:4000, Sigma-Aldrich), were used in the Western blot analysis. The immunoreactive signals were visualized using enhanced chemiluminescence detection. To quantify protein levels, X-ray films were scanned and analyzed using Image J 1.47i software (National Institutes of Health, Bethesda, MD, USA).