Figure 5.

HK2 serves as a coactivator of Nrf2 in regulating XOR expression. A, IL-1β increases XOR levels under conditions of glucose deprivation. siRNA-mediated knockdown of either Nrf2 (B) or HK2 (C) prevents IL-1β–induced XOR expression in glucose-deprived cells as depicted by Western blot analysis. Insets show knockdown efficiency of Nrf2 and HK2 siRNAs. Western blot images are representation of three independent experiments showing similar results. Blots were reprobed for β-actin to establish equivalent loading. Densitometry data of -fold change in XOR expression over control under different treatment conditions normalized to corresponding loading controls are shown. Each data point in the scatter plot denotes -fold change with respect to control from independent experiments (n = 3). D and E, ChIP performed on the region containing the ARE site on XOR promoter in T98G and U87MG glioma cells indicates increased Nrf2 and HK2 binding at the ARE site in IL-1β–treated glucose-deprived cells. F, ChIP assay depicting decreased HK2 binding at XOR promoter upon siRNA-mediated Nrf2 knockdown in IL-1β–treated glucose-deprived cells. Diluted input (5%) was used as a positive control. Relative enrichment was calculated with respect to control levels after correction for background signals. Graphs are representative data of three independent experiments. −G denotes glucose-deprived DMEM. One-way ANOVA (Bonferroni's multiple comparison test) was used for statistical analysis. Error bars represent S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001. G, schematic depiction of the importance of HK2 as a coactivator of Nrf2 in regulation of XOR under inflammatory and metabolic stresses. NSsiRNA, nonspecific siRNA; qPCR, quantitative PCR.