Figure 6

XBP-1 plays an important role in up-regulation of TRAIL-R2 by 2-DG. A, 2-DG activates the UPR in melanoma cells. Mel-RM and MM200 cells were treated with 2-DG (10 μM) for indicated periods. Upper panel: Whole cell lysates were subjected to Western blot analysis. Lower panel: RT-PCR products of XBP-1 mRNA from the cells were digested with Apa-LI for 90 minutes followed by electrophoresis. The longer fragment derived from the active form of XBP1 mRNA and two shorter bands derived from the inactive form are indicated. B, Mel-RM and MM200 cells were transfected with the control, IRE1α, ATF6, or PERK siRNA. Twenty-four hours later, whole cell lysates were subjected to Western blot analysis. C, Mel-RM (left panel) and MM200 (right panel) cells were transfected with the control, IRE1α, ATF6, or PERK siRNA. Twenty-four hours later, cells were treated with 2-DG (10 μM) for 24 hours. The cell surface expression of TRAIL-R2 was measured using flow cytometry. The data in the y axes represent mean fluorescence intensity (MFI). D, Whole cell lysates from Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA were subjected to Western blot analysis. E, Left panel: Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA were treated 2-DG (10 μM) for 24 hours. The cell surface expression of TRAIL-R2 was measured using flow cytometry. Filled histograms: isotype controls; Thick open histograms: TRAIL-R2 expression before treatment; Thin open histograms: TRAIL-R2 expression after treatment with 2-DG (10 μM) for 24 hours. Right panel: Total RNA from Mel-RM and MM200 cells transduced with the control or XBP-1 shRNA treated with 2-DG (10 μM) for 16 hours was isolated and subjected to Real-time PCR analysis for TRAIL-R2. The relative abundance of mRNA expression before treatment was arbitrarily designated as 1. Deficiency in XBP-1 significantly blocked up-regulation of TRAIL-R2 mRNA by 2-DG (p < 0.05). The data shown are either the mean ± SE (C & the right panel of E), or representative (A, B, D, & the left panel of E), of three individual experiments.