Figure 3

TCTP inhibits caspase activation in etoposide-induced cell death. (A) TCTP-induced inhibition of caspase-9, -7, and -3 fragmentation. AdGFP (G)- and adTCTP-GFP (T)-infected cells (MOI, 10) were treated with 20 μM etoposide and the activation of caspases were determined by western blot assay using cleaved form-specific caspase-9, -7, and -3, and PARP antibodies (left panel). Cleavage of procaspase-9 was further assayed using specific antibodies detecting 35- and 37-kDa form of cleaved caspase-9 (right panel). (B) TCTP-induced inhibition of caspase-9 and -3 cleavages under in vitro assays of apoptosome reconstitution. S-100 extracts were incubated with dATP/cyto c to induce the apoptosome formation. Activation of caspase-9 and -3 was detected by western blotting in the presence or absence of TCTP in the reaction. (C) TCTP-induced inhibition of caspase-9 and -3 activities confirmed by inhibitor assay. The specific inhibitor of caspase-9 and -3, Ac-LEHD and Ac-DEVD, respectively, were used. Caspase activity was presented as RFU by using fluorogenic substrates for caspases. Error bar represent SD of two independent experiments. (D) Time- and dose-dependence of TCTP-induced inhibition of caspase-9 and -3 fragmentations. Following dose- and time-dependent incubation with His-tagged TCTP at a time of 30 min and at a dose of 1 μg/ml, respectively, western blot assay was performed to detect caspase activity using anti-Apaf-1, -cleaved caspase-9, -cleaved caspase-3, and -His-specific antibodies.