FIGURE 4.

Depletion of PKN1 increases apoptosis initiated by WNT3A stimulation. A–E, A375 and Mel-624 malignant melanoma cells were transfected with the indicated siRNA oligonucleotides. 48 h later, the cells were treated with medium containing recombinant WNT3A (rWNT3A; 100 ng/ml) or conditioned with WNT3A-secreting (W3ACM) or vehicle-secreting (LCM) L cells. To determine the effects of PKN gene depletion on melanoma cell death, Western blot (WB; A and E) or flow cytometry (B–D) analysis was performed. The abundance of cleaved PARP was used to assay for apoptosis in Western blot studies (A, top panel; E, left panel). Blots from three independent experiments were quantified by densitometry (A, bottom panel; E, right panel). As a second assay for apoptosis, the percentage of A375 (B and C) and Mel-624 (D) cells expressing AnnexinV was calculated. Representative plots (B) and quantification of three independent experiments (C and D) are presented. In A, C, D, and E, error bars represent S.E., and p values (*, p < 0.05; ***, p < 0.001; ****, p < 0.0001) were calculated using two-way ANOVA followed by a Bonferroni post-test. CTRL, control.