Figure 4

Downregulation of the Bim_EL protein in melanoma cells undergoing ER stress is mediated by MEK/ERK signaling. (a) The proteasome inhibitor MG132 reverses downregulation of the Bim_EL in melanoma cells under ER stress. Mel-RM cells with or without pretreatment with the proteasome inhibitor MG132 (10 μM) for 1 h were treated with TM (3 μM) for 24 h. Thirty microgram of total protein of whole-cell lysates were then subjected to western blot analysis of Bim and GAPDH (as a loading control). The data shown are representative of three individual experiments. (b) ER stress accelerates the turnover rate of Bim_EL in melanoma cells. Upper panel: Mel-RM cells were treated with the protein synthesis inhibitor cycloheximide (CHX) (10 μg/ml) with or without the addition of TM (3 μM) for indicated periods. Thirty microgram of total protein of whole-cell lysates were then subjected to western blot analysis of Bim and GAPDH (as a loading control). The data shown are representative of three individual experiments. Lower panel: quantitative expression levels of Bim_EL as shown the upper panel that were normalized to GAPDH. Quantitation of each band was determined using ImageReader LAS-4000. The data shown are representative of three individual experiments. (c) TM phosphorylates Bim_EL in melanoma cells but dephosphorylates it in MCF-7 cells. Mel-RM and MCF-7 (upper panel) and IgR3, Sk-Mel-28, and MM200 (lower panel) cells were treated TM (3 μM) for indicated periods. Thirty microgram of total protein of whole-cell lysates were then subjected to western blot analysis using an antibody that specifically recognizes Bim_EL phosphorylated at Ser69. Western blot analysis of GAPDH was included as a loading control. The data shown are representative of three individual experiments. (d) TG induces phosphorylation of Bim_EL in melanoma cells. Mel-RM and MM200 cells were treated with TG (1 μM) for indicated periods. Thirty microgram of total protein of whole-cell lysates were then subjected to western blot analysis using an antibody that specifically recognizes Bim_EL phosphorylated at Ser69. Western blot analysis of GAPDH was included as a loading control. The data shown are representative of three individual experiments. (e) TM increases an increase in ubiquitination of Bim_EL in Mel-RM melanoma cells, but a decrease in MCF-7 breast cancer cells. Whole-cell lysates from Mel-RM melanoma cells and MCF-7 breast cancer cells with or without treatment with TM (3 μM) for 36 h were subjected to immunoprecipitation using an antibody against Bim. Thirty microgram of total protein of the resulting precipitates were subjected to SDS-PAGE and probed with an antibody against ubiquitin and an antibody against Bim. The data shown are representative of three individual experiments. (f) The MEK inhibitor U0126 inhibits phosphorylation of Bim_EL and increases its expression in melanoma cells undergoing ER stress. Thirty microgram of total protein of whole-cell lysates from Mel-RM cells treated with U0126 (20 μM), TM (3 μM), or U0126 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. (g) Knockdown of MEK1 by siRNA inhibits phosphorylation of Bim_EL and increases its expression in melanoma cells undergoing ER stress. Mel-RM cells were transfected with the control or MEK1 siRNA. Twenty-four hours later, cells were treated with TM (3 μM) for a further 24 h. Thirty microgram of total protein of whole-cell lysates were then subjected to western blot analysis of Bim, MEK1, pERK (pERK), ERK, and GAPDH (as a loading control). The data shown are representative of three individual western blot analyses