Figure 5

XBP-1 mediates upregulation of Ets-1 in melanoma cells under ER stress. (a) Mel-CV and MM200 cells were transfected with the control, IRE1α, ATF6 and PERK shRNA, respectively. Upper panel: whole cell lysates were subjected to western blot analysis. Quantitation of the bands showed that the IRE1α, ATF6 and PERK shRNA inhibited IRE1α, ATF6 and PERK expression by 71 and 70%, 67 and 75%, 76 and 78%, in Mel-CV and MM200 cells, respectively. Lower panel: whole cell lysates from cells treated with or without TM (3 μM) for 16 h were subjected to western blot analysis. (b) Mel-CV and MM200 cells with or without IRE1α and ATF6 knocked down by shRNA, respectively, were treated with TM (3 μM) for 16 h. Total RNA was subjected to qPCR analysis for Mcl-1 mRNA expression. The relative abundance of mRNA expression in cells carrying the control shRNA without treatment with TM was arbitrarily designated as 1. (c) Mel-CV and MM200 cells were transfected with the control and XBP-1 shRNA, respectively. Upper panel: whole cell lysates were subjected to western blot analysis. Quantitation of the bands showed that the XBP-1 shRNA inhibited XBP-1 expression by 82 and 85% in Mel-CV and MM200 cells, respectively. Lower panel: whole cell lysates from cells treated with or without TM (3 μM) for 16 h were subjected to western blot analysis. (d) Mel-CV and MM200 cells with or without pretreatment with the PI3k inhibitor LY294002 (20 μM) for 1 h were treated with TM (3 μM) for a further 16 h. Whole cell lysates were subjected to western blot analysis. (e) MM200 cells were transfected with the control or Akt3 small RNA interference (siRNA). Upper panel: After 24 h, whole cell lysates were subjected to western blot analysis. Quantitation of the bands showed that the Akt3 siRNA inhibited Akt3 expression by 90%. Lower panel: After 24 h, cells were treated with TM (3 μM) for a further 16 h. Whole cell lysates were subjected to western blot analysis. The intensity of the Ets-1 bands was quantitated and the value in cells transfected with the control siRNA without treatment with TM was arbitrarily designated as 1. The relative expression level of Ets-1 in each sample was depicted below the corresponding blot.