Figure 5.

The pentose phosphate pathway is altered in NRF2-knockdown HNSCC cells. (A) GSEA enrichment plots and heat maps of differentially expressed genes belonging to the KEGG PENTOSE PHOSPHATE PATHWAY (PPP) and associated with knockdown of NRF2 in HNSCC cells. (B) GSEA of the TCGA-HNSC dataset showed that NRF2 level was positively correlated with the PPP pathway. (C) GSEA demonstrated that upregulation of the PPP was associated with poor survival of HNSCC according to gene expression and overall survival data in the TCGA-HNSC dataset. (D) Heat-map of relative gene expression, assessed via real-time PCR, in NRF2-knockdown cells compared to the non-targeted negative control. Gene expression in the NRF2-knockdown cells was significantly different (p < 0.05) compared to scramble control and is represented as the average value of three individual experiments per heat map square (siControl = 100%). (E) Total G6PD and TKT protein levels were assessed in NRF2-knockdown HONE-1 and Ca9-22-D1 cells by Western blot. β-Actin was detected as a loading control. (F) G6PD and TKT protein levels were assessed in NRF2-overexpressing DOK cells treated with NRF2 siRNA or non-targeted control. Examination of the changes of G6PD (G) and TKT (H) enzyme activities in NRF2-overexpressing DOK cells treated with NRF2 siRNA or non-targeted control. G6PD (I) and TKT (J) enzyme activity assays were performed on the excised tumors from Ca9-22-D1 xenograft tumor-bearing mice. ChIP-qPCR analysis of G6PD (K) and TKT (L) were performed on HONE-1 and Ca9-22-D1 cells by using normal rabbit IgG or an anti-NRF2 antibody, and the result was normalized to input control values and represented as the fold enrichment relative to the anti-normal rabbit IgG control. All data are expressed as the mean ± S.D. from three individual experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.