Figure 3

Functional analysis. (A) Requirement of both survivin-ΔEx3 BH2 and BIR domains for caspase-3-inhibition. At 48 h after transfection of the indicated plasmids, and after 2 h of TNFα/CHX treatment, fluorescent substrate (Sub) and cellular extracts expressing the different constructs were mixed together and reactions were incubated for 1 h at 37°C, before monitoring fluorescence on a fluoremeter. Results present caspase-3 activity in per cent, each sample standardised to the noninduced cellular extracts, as requested by the manufacturer. (B) Both BH2 and BIR domains of survivin-ΔEx3 are essential for its anti-apoptotic function. At 48 h after transfection, in HeLa cells transfected with the indicated expression constructs and exposed to TNFα/cycloheximide during 2 h, MitoTracker was used to measure the loss of mitochondrial membrane potential (ΔΨm). Inhibition percentage was calculated as follows: (% apoptosis in vector-transfected cells)−(% apoptosis in the indicated DNA-transfected cells)/(% apoptosis in vector-transfected cells), where % apoptosis is the percentage of apoptotic cells relative to total cells. (C) Survivin-ΔEx3 can inhibit Bax-induced cytochrome c translocation. Subcellular fractionation assays were performed with 293T cells expressing the different constructs. Cytosolic fraction (C) and mitochondrial fractions (Mito) were blotted with an anti-cytochrome c antibody.