Figure 4

Ets-1 is responsible for transcriptional upregulation of Mcl-1 by ER stress in melanoma cells. (a) Mel-CV and MM200 cells were transfected with the control and Ets-1 small RNA interference (siRNA), respectively. Left panel: After 24 h, cells were treated with TM (3 μM) for a further 16 h. Total RNA was subjected to qPCR analysis for Ets-1 mRNA expression. The relative abundance of mRNA expression in parental cells was arbitrarily designated as 1. Right panel: After 24 h, whole cell lysates were subjected to western blot analysis. Quantitation of the bands showed that the Ets-1 siRNA inhibited Ets-1 expression by 78 and 82% in Mel-CV and MM200 cells, respectively. (b) Upper panel: Mel-CV cells were co-transfected with the control and Ets-1 siRNA and the pGL3-basic-based luciferase reporter constructs, pGL3-vector and pGL3-Mcl-1−205/+10 of the Mcl-1 promoter, respectively. After 24 h, cells were treated with the vehicle control (control) or TM (3 μM) for a further 16 h, followed by measurement of the luciferase activity. Lower panel: Mel-CV and MM200 cells were transfected with the control and Ets-1 siRNA. After 24 h, cells were treated with TM (3 μM) for a further 16 h. Total RNA was subjected to qPCR analysis for Mcl-1 mRNA expression. The relative abundance of mRNA expression in cells transfected with the control siRNA without treatment with TM was arbitrarily designated as 1. (c) Mel-CV and MM200 cells were transiently transfected with the vector alone or complimentary DNA encoding Ets-1. After 24 h, whole cell lysates were subjected to western blot analysis. (d) Upper panel: Mel-CV and MM200 cells co-transfected with complimentary DNA encoding Ets-1 and the pGL3-basic-based luciferase reporter constructs, pGL3-vector and pGL3-Mcl-1−205/+10 of the Mcl-1 promoter, respectively. After 24 h, the luciferase activity was measured. Lower panel: Mel-CV and MM200 cells were transiently transfected with the vector alone or complimentary DNA encoding Ets-1. After 24 h, total RNA was subjected to qPCR analysis for Mcl-1 mRNA expression. The relative abundance of mRNA expression in cells with mock transfection was arbitrarily designated as 1. Bars, s.e. (n=3).