FIGURE 2.

WTX promotes NRF2-dependent transcription. A, two nonoverlapping siRNAs reduce WTX transcript levels. HEK293T cells were transfected with the indicated siRNAs. Total RNA was isolated 48 h after transfection, and relative transcript levels were quantified as the ratio compared with β-actin transcripts. B, WTX is required for activation of an ARE-luciferase reporter. HEK293T cells stably expressing an ARE-driven firefly-luciferase reporter as well as a control CMV-driven Renilla luciferase reporter were transfected with siRNAs and either DMSO or tBHQ. Relative levels of ARE-driven firefly luciferase compared with control CMV-driven Renilla luciferase are plotted. Error bars represent means ± S.D. of three technical replicates. Data are representative of three biological replicates. C, overexpression of WTX activates an ARE-luciferase reporter. HEK293T cells were transfected with the ARE-firefly luciferase reporter, a control CMV Renilla luciferase control reporter, and the indicated FLAG-tagged DNA constructs. Cells were treated with tBHQ, and luciferase levels were quantified as in B. D, loss of WTX inhibits transcription of endogenous NQO1. H1299 cells expressing an NQO1-YFP fusion protein under control of the endogenous promoter were transfected with the indicated siRNAs and treated with either DMSO or tBHQ. Relative YFP fluorescence is plotted. Error bars represent means ± S.D. of four technical replicates. Data are representative of three biological replicates. *, p < 0.02 when comparing WTX siRNA with control. E, loss of WTX increases the cellular sensitivity to etoposide. HEK293T cells were transfected with the indicated siRNAs and treated with etoposide. Cell viability was determined by measuring the metabolic activity of mitochondria (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay). *, p < 0.03 when comparing NRF2 and WTX siRNAs with control.