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. 2021 Jan 20;13(2):128–140. doi: 10.1093/jmcb/mjaa079

Figure 3.

Figure 3

Upregulation of NRF1 transcriptionally activates p65-coding gene RELA in L132 cells exposed to CSE or LPS. (A‒J) L132 cells were transfected with NRF1 siRNA (si-NRF1) or control siRNA (si-CTRL) for 24 h. Cells were treated with 2% CSE for 42 h followed with 1 μg/ml LPS or 10 ng/ml PAM3CSK4 for 6 h. (A‒C) Protein levels of NRF1 and p65 were determined by western blotting. (D and E) mRNA expression levels of NRF1 and RELA were determined by qRT-PCR. (F) Enriched DNA fragments by NRF1 were pulled down for ChIP–seq. Target genes were analyzed by MAnorm software. (G‒J) RELA promoter fragment enrichment of NRF1 was determined by ChIP–qPCR. CYCS, the target gene of NRF1, served as a positive control. (K) HEK293T cells were co-transfected with NRF1 siRNA and pGL3-RELA for 48 h. The pRL-TK vector was co-transfected to normalize transfection efficiencies. (L‒N) L132 cells were pretreated with Bay11-7082 for 24 h and exposed to CSE for 42 h followed with 6-h LPS treatment. Cell viability (L) as well as protein levels (M) and mRNA expression levels of NRF1 and p65 (N) were determined. mean ± SD, n.s. means no significance, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 by one-way ANOVA.