Apoptosis induction

A total of 100 ng/mL TNF-related apoptosis-inducing ligand (TRAIL) was used to induce rapid extrinsic apoptosis in HCT116 cells. Where indicated, HCT116 cells were pre-treated for 1 hr with 40 μM caspase Inhibitor Z-VAD-FMK (zVAD) or 25 μM ivermectin before TRAIL treatment. Extrinsic apoptosis induction was confirmed by observing CASP8 and CASP3 cleavage on western blots. CASP3/8 cleavage was quantified by disappearance in intensity of the full-sized band normalized to TUBA1A or VCL using Bio-Rad ImageLab software. Intrinsic apoptosis was induced in HeLa cells with a 4 hr treatment of 10 μM raptinal, with or without 1 hr pre-treatment with 20 μM zVAD. Induction was confirmed by evidence of cytochrome c release into the cytoplasm with cell fractionation and western blot. ‘Mock’ treatments consisted of an equal volume of vehicle used to dissolve each reagent: TRAIL storage and dilution buffer, DMSO, and ethanol for TRAIL, zVAD, and ivermectin, respectively.