Figure 4
Vesicular overexpressed in cancer prosurvival protein 1 (VOPP1) knockdown-induced cell death is the result of the activation of apoptosis. (a) Results of enzymatic assays for caspase-3/7 in small interfering (si)RNA-mediated knockdown experiments in the squamous cell carcinoma (SCC)-9 cell line are shown in the upper panel. The data shown is the average of two independent experiments, conducted in at least quadruplicate each. Only 72-h time-point differences were significant (P<0.05 marked by asterisks, Student's t-test). The lower panel shows pro-caspase-3 cleavage, followed by western blot at the same time points from an independent experiment. Both of these results indicate that a significant caspase-3/7 activation does not occur initially occur when VOPP1 expression is first reduced, but rather occurs at 72 h post-siRNA transfection. (b) Results of the enzymatic assays for caspase-3/7 activation in siRNA-mediated knockdown of VOPP1 are shown in multiple cell lines. The data represent independent experiments conducted in triplicate for each cell line noted. All cell lines show significant (P<0.05 marked by asterisks, Student's t-test) caspase-3/7 activation only with VOPP1 targeting siRNA in relation to control at 72 h after transfection, which is coincident with the time point wherein cell viability was observed to decrease in earlier experiments. (c) Results of enzymatic assays for caspase-9 in siRNA-mediated knockdown experiments in SCC-9 and HeLa cell lines are shown. Paclitaxel is included as a positive control for the induction of apoptosis via the intrinsic pathway. The data indicate that caspase-9 activation by VOPP1 knockdown is observed 48 h after siRNA transfection. Data are from independent experiments conducted in triplicate for each condition noted. All error bars represent 95% confidence intervals with asterisks denoting significant differences for VOPP1 targeting siRNAs relative to control, P<0.05 from a Student's t-test without assuming equal variances.
