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. 2015 Apr 20;6(18):16168–16182. doi: 10.18632/oncotarget.3753

Figure 2. Crucial roles of NFκB in ITLN1-mediated regulation of HNF4α expression in gastric cancer cells.

Figure 2

A. one potential binding site of NFκB was noted within the HNF4α promoter, locating at bases 508-522 upstream the transcription start site (TSS). ChIP and qPCR assays indicated the endogenous binding of NFκB-p65 on −606/−409 bp region, but not on −339/−158 region, of the HNF4α promoter in SGC-7901 and AGS cells. B. ChIP and qPCR assays indicated that over-expression or knockdown of ITLN1 decreased and increased the binding of NFκB-p65 on the HNF4α promoter, which was rescued by over-expression and knockdown of NFκB-p65 (*P < 0.01 vs. mock or sh-Scb). C. dual-luciferase assay indicated that stable transfection of ITLN1 or sh-ITLN1 into gastric cancer cells facilitated or suppressed the promoter activity of HNF4α than those in mock or sh-Scb-transfected cells, respectively, which was rescued by transfection of NFκB-p65 or sh-NFκB-p65. In addition, mutation of NFκB binding site abolished these effects (*P < 0.01 vs. sh-Scb or mock). D. western blot assay indicated that transfection of NFκB-p65 prevented the gastric cancer cells from ITLN1-mediated up-regulation of HNF4α. E. western blot assay indicated that knockdown of NFκB-p65 prevented gastric cancer cells from sh-ITLN1- repressed expression of HNF4α.