Figure 7.
PI3K inhibition induces cell cycle arrest in melanoma cells and enhances cytotoxicity of BRAFV600E inhibition. Melanoma cell lines were treated with Pi-103 and/or PLX4720. (A) Samples were kept in medium containing Pi-103 for 7 d and fixed and stained with crystal violet. (B) Samples were analyzed for BrdU incorporation 4 d after treatment with increasing concentrations of Pi-103 (0.25, 0.5, 1, and 2 μM). Error bars represent SEM (standard error of the mean) of three independent experiments. For 453A0 melanoma cells, a representative experiment is shown. (C) Samples were analyzed by Western blotting for p15INK4B 2 d after treatment with 0.25 μM Pi103 and/or 1 μM PLX4720. β-Actin served as a loading control. (D) Melanoma cell lines were treated with 0.5 μM Pi-103 and/or 2 μM PLX4720 for 4 h. Samples were analyzed by Western blotting for the indicated proteins. β-Actin served as a loading control. (E) Melanoma cell lines were treated with a dilution series of Pi-103 either alone or in combination with the BRAFV600E inhibitor PLX4720 at a concentration of 3 μM (D10) or 1 μM (453A0) for 3 d. Total cell numbers were determined with a cell titer blue assay. The Y-axis represents the percentage of living cells. (F) Cells were treated with 0.25 μM Pi-103 and/or 1 μM PLX4720 for 1 d (D10) or 3 d (453A0). Samples were analyzed by Western blotting for cleaved caspase 3. Apoptotic cells in the supernatant were included in the analysis. β-Actin served as a loading control. (G) Cells were treated with a dilution series of PLX4720 either alone or in combination with 0.5 μM Pi-103. Total cell numbers were determined with a cell titer blue assay. The Y-axis represents the percentage of living cells.