Figure 4.
VHL-Deficient B Cells Exhibit Exacerbated Caspase-8 Activation
(A) Flow cytometric detection of caspase-3 activation in VHL-deficient B cells. VHL-deficient and WT B cells were cultured for 2 or 4 h and stained for activated caspase-3.
(B) Survival of VHL-deficient B cells after treatment with pan-caspase inhibitor Z-VAD. VHL-deficient B cells were cultured in medium alone or in the presence of Z-VAD (10μM) for 8 h followed by flow cytometry to detect Annexin V− live cells. Numbers indicate percentage of Annexin V− live cells (mean ± SD, n = 5).
(C) Survival and caspase-3 activation in B cells under normoxia and hypoxia. WT B cells were cultured under normoxia (21% O2) or hypoxia (1% O2) for 16 h followed by flow cytometry to detect Annexin V− live cells and caspase-3 activation. Numbers indicate percentage of Annexin V− live cells (upper panel, mean ± SD, n = 5) or activated caspase-3+ cells (lower panel, mean ± SD, n = 5).
(D) Immunoblotting of caspase-8 activation in VHL-deficient B cells. Whole-cell lysates of VHL-deficient and WT B cells were subjected to immunoblotting to detect pro- and cleaved-caspase-8. β-Actin was used as loading control.
(E) Survival of VHL-deficient B cells after treatment with pan-caspase inhibitor Z-VAD (10μM), caspase-8 inhibitor Z-IETD (10μM) or caspase-9 inhibitor Z-LEHD (10μM) for 8 h. Numbers indicate percentage of Annexin V− live cells (mean ± SD, n = 5).
(F) DISC formation in VHL-deficient B cells. VHL-deficient and WT B cells were lysed and subjected to immunoprecipitation with anti-Fas antibody. Fas-associated caspase-8 and FADD were detected by immunoblotting subsequently.
Data are representative of 4 (A) or 5 (D, F) experiments. See also Figures S4 and S7.