Caspase 8

MTT, acridine orange, di-2-pyridylketone, pifithrin-α, and monodansylcadaverine (MDC) were purchased from Sigma-Aldrich. LC3 antibody was obtained from Proteintech Group (Wuhan, China), caspase-8, GAPDH, bax, cytochrome c, and bcl-2 were purchased from Boster (Wuhan, China). RPMI 1640 and fetal bovine serum were purchased from Zhejiang Tianhang Biological Technology Co. Ltd.

Cells were harvested and then lysed using 200‐μL ice‐cold RIPA lysis buffer (KeyGEN BioTECH, KGP702‐100) consisting of 1 mmol/L PMSF and protease inhibitor cocktail. A commercially available BCA protein assay kit (KeyGEN BioTECH, KGP902) was used to quantify proteins in the lysate. Cell lysates consisting of 100‐µg protein were loaded onto a sodium dodecyl sulfate polyacrylamide gel with an 8%‐12% gradient. After transferring the proteins to polyvinylidene membranes, they were detected using immunoblotting methods with antibodies against the relevant proteins, as follows: antibody to β‐actin (BM0627) was obtained from Boster (Wuhan, China); caspase‐8 (CST, #4927), caspase‐9 (CST, #9504), AMPKα (CST, #2532), Phospho‐AMPKα (Thr172) (CST, #2531), p44/42 MAPK (Erk1/2) (CST, #9102), Phospho‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (CST, #4370), IRS‐1 (CST, #2382), and Phospho‐IRS‐1 (Ser307) (CST, #2381) were procured from Cell Signaling Technology; COX IV (AB3119), Bcl‐2 (AB182858), Caspase‐3 (AB13847), AIF (AB1998), and cytochrome C (Cyt C, AB90529) were procured from Abcam, Ltd.; and Phospho‐IRS‐1 (Tyr628) mouse/(Tyr632) (09‐433) was obtained from Sigma‐Aldrich. Protein bands were visualized using a commercially available enhanced chemiluminescence assay kit (Bio‐Rad) according to the manufacturer's instructions.