For active Caspase-3 staining, HeLa cells were cultured in 35 mm glass base dishes (Iwaki) and, when indicated, transfected with either empty pBI-CMEF, pBI-CMEF-FLAG-Bcl2l10, pBI-CMEF-FLAG-IRBITS68A or pBI-CMEF-FLAG-IRBITS68A-FLAG-Bcl2l10. 24 hr after transfection, cells were treated with DMSO (1/1000, Sigma-Aldrich) for 24 hr, 1 µM staurosporine (LKT Laboratories) for 4 hr, 2 µM thapsigargin (Calbiochem) for 24 hr or 20 µM tunicamycin (Sigma-Aldrich) for 24 hr and then stained using the Image-iT LIVE Green Caspase-3 and −7 Detection Kit (ThermoFisher Scientific) according to the manufacturer's instructions. Images were acquired with a fluorescence microscope (Biozero BZ-8100, Keyence).
For western blot analysis of apoptosis, cells were transfected, when indicated, with empty pHM6 or pHM6-IRBIT and treated, 24 hr after transfection, with DMSO (1/1000, Sigma-Aldrich) for 24 hr, 1 µM staurosporine (LKT Laboratories) for 4 hr, 2 µM thapsigargin (Calbiochem) for 24 hr or 20 µM tunicamycin (Sigma-Aldrich) for 24 hr. After treatment, cells were lysed in TNE buffer, the protein concentration of each sample was determined using the Bradford assay (Bio-Rad) and equivalent amounts of protein (10 μg) were analyzed by immunoblotting with appropriate antibodies.
For western blot analysis of apoptosis, cells were transfected, when indicated, with empty pHM6 or pHM6-IRBIT and treated, 24 hr after transfection, with DMSO (1/1000, Sigma-Aldrich) for 24 hr, 1 µM staurosporine (LKT Laboratories) for 4 hr, 2 µM thapsigargin (Calbiochem) for 24 hr or 20 µM tunicamycin (Sigma-Aldrich) for 24 hr. After treatment, cells were lysed in TNE buffer, the protein concentration of each sample was determined using the Bradford assay (Bio-Rad) and equivalent amounts of protein (10 μg) were analyzed by immunoblotting with appropriate antibodies.