Figure 3.
Abrogation of BRAFV600E-induced senescence by PTEN depletion is dose-dependent. Primary human melanocytes stably expressing sh-p16INK4A and either control or sh-PTEN (#1) were transduced with control or BRAFV600E-encoding lentivirus and selected for integration of the construct. Empty vectors were used as controls. (A) Samples were analyzed by Western blot for protein expression as indicated. β-Actin served as a loading control. (B) Samples were analyzed for BrdU incorporation 15 d post-infection. Data are represented as mean ± SD (one-way ANOVA/Bonferroni's multiple comparison test; P < 0.05). (C) Samples were fixed and analyzed for SA-β-Gal activity 15 d post-infection. Data are represented as percentage of cells ± SD (one-way ANOVA/Tukey's multiple comparison). For both B and C, BRAFV600E sh-PTEN1:10 and BRAFV600E sh-PTEN1:25 samples are significantly different from BRAFV600E controls (P < 0.05). (D) Samples were seeded at equal densities and fixed and stained 15 d post-infection. Crystal violet staining was extracted and quantified; similar results were obtained in duplicate experiments.