Fig 1: O-GlcNAc NRF1 increases the pool of gene transcription to protect cells against genotoxicity.a Effect of NRF1 O-GlcNAc modification on the indicated gene transcription levels in ADR cells. The gene mRNA levels in ADR cells expressing Flag-WT-NRF1 or Flag-AA-NRF1 were analyzed by quantitative PCR (qPCR). b Left panel: IGV tracks showing the signals at the promoter regions of the representative genes. Right panel: Validation of O-GlcNAc NRF1 binding peaks by ChIP-qPCR. qPCR amplification was performed. Each bar represents the fold enrichment of binding relative to the input. IgG and random primers that could not specifically bind the indicated gene promoter regions (off target) were used as negative controls. Mock, cells transfected with empty pCMVPuro64 vector. c O-GlcNAc inhibition reduces the transcriptional activity of NRF1. 293T cells were transfected with a reporter vector consisting of luciferase cDNA fused to the NSMCE2 promoter. The pGL3-basic vector (Mock) was used as a control. d ADR cells were transfected with NSMCE2 siRNA (siNSMCE2) or scrambled siRNA (siScr) and treated with increasing doses of Adm for 48 h. The cell viability was then assessed. Representative images of cell viability determined by crystal violet staining are shown. Results were reproduced in two biologically independent experiments. The protein levels of NSMCE2 were monitored by immunoblotting. All blots are representative of at least two biologically independent experiments. For (a-c), the data are presented as the means ± SEM., (d) replicates are represented. (a–d) n = 3 biologically independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001 (two-sided unpaired Student’s t-test). p values: 0.010099, 0.001375, 0.046814, 0.020148 (a); 0.010191, 0.000117, 0.000159, 0.001032 (b); 0.0065 (WT-NRF1 vs. WT-NRF1 L01), 0.0002 (WT-NRF1 vs. AA-NRF1), 0.0000858 (WT-NRF1 vs. AA-NRF1 L01) (c); 0.001575 (d). a–d Experiments were repeated independently two times with similar results. Source Data are provided as a Source Data file.