Figure 5: Pro-apoptotic effects of climacostol in melanoma cells depend on p53 activity.

(a,b) B16-F10 cells were cultured in the presence of 30 μg/ml climacostol or vehicle (control) for 24 h. (a) Western blot analysis of p53 expression. Vinculin was used as the internal standard; Lower panel: densitometry analysis of p53 expression (n = 3) *p < 0.001 vs control. (b) mRNA levels of p53 target genes p21, Noxa and Puma, as measured by real-time PCR. Each histogram represent the results obtained from 3 independent experiments. Data are expressed as the fold change over control (set as 1). *p < 0.001 and **p < 0.0001 vs the respective control. (c,d) B16-F10 cells were transiently transfected for 48 h with a p53-specifc (p53 siRNA) and a non-targeting siRNA (nt siRNA). Cells were then treated for 24 h with 30 μg/ml climacostol or vehicle (control). (c) Western blot analysis of p53 expression. Vinculin, HSP60, LDH-A, and GAPDH were used as internal standards and to test possible off-target effects of siRNA transfection (n = 3). (d) Evaluation by flow cytometry of Annexin V/PI staining (Annexin V⁺/PI⁻ + Annexin V⁺/PI⁺ fraction; the Annexin V⁻/PI⁺ fraction, regarded as necrotic stage, was excluded). Data are expressed as fold change over the respective control. Each histogram represent the results obtained from 3 independent experiments. *p < 0.001 vs nt siRNA.