Figure 5

Downregulation of the Bim_EL protein in melanoma cells under ER stress is associated with reduction in PP2A activity that is, at least in part, due to downregulation of the PP2A catalytic C subunit (PP2A-C). (a) Pharmacological activation of PP2A reverses downregulation of Bim_EL by ER stress in melanoma cells. Left panel: 30 μg of total protein of whole-cell lysates from Mel-RM melanoma cells or MCF-7 breast cancer cells treated with the pharmacological PP2A activator FTY720 (2.5 μM), TM (3 μM), or FTY720 plus TM for 24 h were subjected to western blot analysis of Bim, pERK, ERK, and GAPDH (as a loading control). The data shown are representative of three individual experiments. Right panel: Mel-RM cells were treated with FTY720 (2.5 μM), TM (3 μM), or FTY720 plus TM for 48 h before apoptosis was measured by the PI method using flow cytometry. The data shown are the mean±S.E. of three individual experiments. (b) Inhibition of PP2A further promotes phosphorylation of Bim_EL and increases phosphorylation of ERK in Mel-RM cells under ER stress. Thirty microgram of total protein of whole-cell lysates from Mel-RM melanoma cells or MCF-7 breast cancer cells treated with the OA (50 nM), TM (3 μM), or OA plus TM for 24 h were subjected to western blot analysis of Bim, pERK, ERK, and GAPDH (as a loading control). The data shown are representative of three individual experiments. (c) Inhibition of PP2A further promotes phosphorylation of Bim_EL in Sk-Mel-28, IgR3, and MM200 melanoma cells. Thirty microgram of total protein of whole-cell lysates from Sk-Mel-28, IgR3, and MM200 cells treated with the OA (50 nM), TM (3 μM), or OA plus TM for 24 h were subjected to western blot analysis of Bim and GAPDH (as a loading control). The data shown are representative of three individual experiments. (d) ER stress reduces PP2A activity in Mel-RM melanoma cells, but increases PP2A activity in MCF-7 breast cancer cells. Mel-RM cells and MCF-7 cells were treated with TM (3 μM) for indicated periods before the phosphatase activity of PP2A was quantitated using a PP2A-C immunoprecipitation phosphatase assay kit. The PP2A phosphatase activity in cells without treatment was arbitrarily designated as 1. The data shown are the mean±S.E. of three individual experiments. (e) ER stress downregulates PP2A-C in melanoma cells but upregulates its expression in MCF-7 breast cancer cells. Upper panel: Thirty microgram of total protein of whole-cell lysates from Mel-RM melanoma cells or MCF-7 breast cancer cells treated with TM (3 μM) for indicated periods were subjected to western blot analysis of PP2A-C and GAPDH (as a loading control). The data shown are representative of three individual experiments. Lower panel: Thirty microgram of total protein of whole-cell lysates from IgR3 and MM200 melanoma cells treated with TM (3 μM) for 24 h were subjected to western blot analysis of PP2A-C and GAPDH (as a loading control). The data shown are representative of three individual experiments. (f) Overexpression of PP2A-C upregulates Bim_EL and decrease ERK phosphorylation in melanoma cells. Mel-RM cells were transiently transfected with vector alone or cDNA encoding PP2A-C. Twenty-four hours later, cells were treated with TM (3 μM) or TG (1 μM) for a further 24 h. Thirty microgram of total protein of whole-cell lysates were then subjected to western blot analysis of Bim, PP2A-C, pERK, ERK, and GAPDH (as a loading control). The data shown are representative of three individual western blot analyses. (g) Overexpression of PP2A-C causes increased PP2A activity in melanoma cells with or without undergoing ER stress. Mel-RM cells were transiently transfected with vector alone or cDNA encoding PP2A-C. Twenty-four hours later, cells were treated with TM (3 μM) for a further 24 h. The phosphatase activity of PP2A was then quantitated using a PP2A-C immunoprecipitation phosphatase assay kit. The PP2A phosphatase activity in cells transfected with vector alone without TM treatment was arbitrarily designated as 1. The data shown are the mean±S.E. of three individual experiments