Figure 2.
MDM2 antagonism induces mitochondrial-mediated apoptosis in senescent melanoma cells. (A) Western Blot analysis of p53 levels and expression of p53 target genes after 5 days of treatment with (−)-Nutlin-3 (10µM), MLN8237 (1µM), combination of both, or vehicle. Numbers are average β-actin-normalized densitometry values from 3 independent experiments. (B) Analysis of apoptosis using Annexin V-FITC and PI staining. SK-Mel5 cells were treated for 5 days as in A. Bottom panel shows the percentages of apoptotic cells per random microscope field. (C) Mitochondrial membrane potential in cells treated as described in A was analyzed using JC-1 dual emission dye. Fluorescence of cells in red (polarized mitochondria) and green (loss of mitochondrial membrane potential) channels were evaluated. Graph shows relative green to red signal ratios in random fields. (D) IF staining of AIF in SK-Mel5 cells treated as described in A. Bottom panel shows the percentages of cells with nuclear AIF staining in random fields. Kruskal-Wallis test with Dunn’s multiple comparison was applied. Experiments were performed at least 3 times with consistent results. Images were quantified with ImageJ software.