Figure 3

ER stress induces transcriptional upregulation of Bim in melanoma cells. (a) TM induces sustained upregulation of Bim mRNA in melanoma cells. Mel-RM melanoma cells and MCF-7 breast cancer cells were treated with TM (3 μM) for indicated periods before total RNA was isolated and subjected to qPCR analysis for the Bim mRNA expression. The relative abundance of the Bim mRNA in each cell line before treatment was arbitrarily designated as 1. The data shown are the mean±S.E. of three individual experiments. (b) CHOP is required for transcriptional upregulation of Bim by ER stress in melanoma cells. Upper panel: Mel-RM melanoma cells and MCF-7 breast cancer cells were transfected with the control or CHOP siRNA. Twenty-four hours later, cells were treated with TM (3 μM) for a further 6 h. Thirty microgram of total protein of whole-cell lysates were then subjected to western blot analysis of CHOP and GAPDH (as a loading control). The data shown are representative of three individual western blot analyses. Lower panel: Mel-RM melanoma cells and MCF-7 breast cancer cells were transfected with the control or CHOP siRNA. Twenty-four hours later, cells were treated with TM (3 μM) for a further 36 h before total RNA was isolated and subjected to qPCR analysis for the Bim mRNA expression. The relative abundance of the Bim mRNA in each cell line transfected with the control siRNA without treatment was arbitrarily designated as 1. The data shown are the mean±S.E. of three individual experiments. (c) The general transcription inhibitor actinomycin D (Act.D) efficiently blocks transcriptional upregulation of Bim in Mel-RM melanoma cells and MCF-7 breast cancer cells. Mel-RM and MCF-7 cells were treated with Act.D (100 ng/ml) for 1 h before the addition of TM (3 μM) for a further 24 h. Total RNA was then isolated and subjected to qPCR analysis for the Bim mRNA expression. The relative abundance of the Bim mRNA in each cell line before treatment was arbitrarily designated as 1. The data shown are the mean±S.E. of three individual experiments. (d) Overexpression of CHOP induces upregulation of the Bim mRNA and the Bim_EL protein in melanoma cells. Left panel: Mel-RM.CHOP cells that carried a lentivirus-based 4-OHT-responsive inducible CHOP expression system were treated with 4-OHT (10 nM) for indicated periods. Thirty microgram of total protein of whole-cell lysates were then subjected to western blot analysis of CHOP, Bim, and GAPDH (as a loading control). The data shown are representative of three individual western blot analyses. Right panel: Mel-RM.CHOP cells that carried a lentivirus-based 4-OHT-responsive inducible CHOP expression system were treated with 4-OHT (10 nM) for indicated periods. Total RNA was isolated and subjected to qPCR analysis for the Bim mRNA expression. The relative abundance of the Bim mRNA in cells without 4-OHT treatment was arbitrarily designated as 1. The data shown are the mean±S.E. of three individual experiments. (e) Overexpression of CHOP induces apoptosis in melanoma cells. Mel-RM.CHOP cells that carried a lentivirus-based 4-OHT-responsive inducible CHOP expression system were treated with 4-OHT (10 nM) 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