Slc7a11

Twenty-five µg of protein extract was separated by SDS-PAGE (8% gels) and transferred to nitrocellulose membranes (GE Healthcare, Freiburg, Germany) by semidry electroblotting. The primary antibodies used were against SLC7A11 (rabbit mAb, clone D2M7A, 1:1000, Cell Signaling, Frankfurt, Germany), HIST1H3B (rabbit pAb, PA5-111876, 1:1000, Thermo Fisher Scientific), HK2 (rabbit mAb, clone C64G5, 1:1000, Cell Signaling), PPARGC1A (mouse mAb, clone 1C1B2, 1:3000, Proteintech, Manchester, UK), ALDH6A1 (rabbit pAb, 20452-1-AP, 1:6000, Proteintech), MARC2 (rabbit pAb, 24782-1-AP, 1:1000, Proteintech), SLC47A1 (rabbit mAb, clone D4C62, 1:500, Cell Signaling), and GAPDH (rabbit mAb, clone 14C10, 1:10,000, Cell Signaling). Secondary horseradish peroxidase-conjugated antibodies against rabbit or mouse were purchased from Jackson ImmunoResearch (Suffolk, UK) and used at a concentration of 1:5000. Protein bands were detected by enhanced chemiluminescence in an LAS-4000 chemiluminescence detection system (GE Healthcare, Munich, Germany).

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).

atRAL, Fer-1 (catalog no. SML0583), DFO (catalog no. D9533), 4',6-diamidino-2-phenylindole (DAPI) and Hoechst 33342 were purchased from Sigma-Aldrich (Saint Louis, MO, USA). 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and BODIPY 581/591 C11 (C11-BODIPY) were obtained from ThermoFisher Scientific (Eugene, OR, USA). Mitotracker Red CMXRos and TRIeasy total RNA extraction reagent were obtained from Yeasen (Shanghai, China). Antibodies against COX2 (catalog no. 12282S), SLC7A11 (catalog no. 98051S), cleaved caspase-3 (catalog no. 9664S and 9661S), and GAPDH (catalog no. 5174S) were provided by Cell Signaling Technology (Danvers, MA, USA). Anti-GPX4 (catalog no. ab125066), anti-ACSL4 (catalog no. ab155282), and anti-acrolein (catalog no. ab48501) were purchased from Abcam (South Cambs, England, UK). Anti-γH2AX (catalog no. 05-636) was purchased from Millipore (Billerica, MA, USA). Anti-γH2AX (catalog no. NB100-384) was provided by Novus Biologicals (Littleton, CO, USA).