Fig. 3: NFκB regulates ONECUT2 protein expression at the transcription level.

A, B In AGS and NCI-N87 GC cells, transfection with 1 or 2 μg of p65 overexpression plasmid resulted in increased p-NFκB protein levels (s536) and increased ONECUT2 protein and mRNA (n = 3). C Protein levels of p-NFκB (s536) and ONECUT2, and ONECUT2 mRNA levels, increased over time with prolonged activation of the NFκB pathway in AGS GC cells (cells were co-cultured with 100 ng/ml TNF-α protein for 3 and 6 h to activate the NFκB pathway) (n = 3). D Protein levels of p-NFκB (s536) and ONECUT2, and ONECUT2 mRNA levels decreased over time with prolonged inhibition of the NFκB pathway in NCI-N87 GC cells (cells were treated with 5 ng/ml BAY protein to inhibit the NFκB pathway for 3 and 6 h) (n = 3). E Schematic representation of two predicted NFκB binding sites for ONECUT2 transcription according to JASPAR. F Dual-luciferase reporter assays showing that HP infection stimulated ONECUT2 promoter transcription in AGS and NCI-N87 cells. G Overexpression of NFκB in AGS and NCI-N87 cells was shown to stimulate transcriptional activity of the ONECUT2 promoter using dual-luciferase reporter assays. H Dual-luciferase reporter assays revealed that activation of NFκB by TNF-α could stimulate transcriptional activity from the ONECUT2 promoter in AGS and NCI-N87 cells. **P < 0.01; ****P < 0.0001.