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. 2021 Aug 5;29(1):147–155. doi: 10.1038/s41418-021-00843-7

Fig. 1. Role of caspase-8 upon proteasome inhibition.

Fig. 1

A Parental HCT-116 (par) and HCT-116 (Bax/Bak)−/− cells were treated with intrinsic apoptosis triggers (100 µg/ml 5-fluorouracil (5-FU), 40 µM cisplatin, 3 µM tunicamycin (tunica), 100 nM bortezomib) as indicated. Cell death was measured by PI uptake. Data are means ± sd from triplicate samples of a representative experiment (*p < 0.05, multiple t-test). Immunoblot insert confirms loss of Bax and Bak in HCT-116 (Bax/Bak)−/− cells. β-actin served as loading control. B Cell death (PI uptake) upon treatment with alternative proteasome inhibitors (100 nM epoxomicin (epoxo) or 10 µM MG132) for 48 h. Data are means ± sd from triplicate samples (p < 0.05, multiple comparisons t-test). C Cells were treated for 48 h as in (A), and cleavage of procaspase-8 and procaspase-3 was analyzed by western blotting. β-actin served as a loading control. Solvent, DMSO control. *Unspecific bands. D Cells were treated for 24 h with 100 nM bortezomib as indicated and cleaved caspase-9 and PARP cleavage were analyzed by western blotting. Tubulin served as a loading control. E Flow cytometric quantification of IETDase activation in HCT-116 (Bax/Bak)−/− cells treated with 100 nM bortezomib, with or without 20 µM z-VAD-fmk for 24 h or 48 h. IETDase activation was measured using a CFP-IETD-YFP FRET probe. *p < 0.05, ANOVA followed by Sidak’s multiple comparisons test. F Quantification of IETD probe cleavage in HCT-116 (Bax/Bak)−/− cells. Cells were transfected with scrambled siRNA or siRNA directed against procaspase-8 (sicasp-8) and treated with 100 nM bortezomib (±50 µM z-VAD-fmk) 24 h after transfection. *p < 0.05 (t-tests). G Bid and procaspase-3 cleavage were studied in HCT-116 (Bax/Bak)−/− cells transfected as indicated and treated with 100 nM bortezomib. Porin and β-actin served as loading controls. *Unspecific bands.