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. 2016 Oct 18;291(49):25553–25566. doi: 10.1074/jbc.M116.751453

FIGURE 5.

FIGURE 5.

OGG1-dependent gene expression in TNFα-exposed cells. A and B, OGG1-dependent gene expression from TNF/Tnf promoters. Left panels, parallel cultures of cells were transfected with control siRNA (□) or siRNA to OGG1 (■), and cells were exposed to TNFα (20 ng/ml) for 30 min. TNF mRNA levels were determined by RT-PCR. A and B, insets, expression of OGG1 at protein levels in cells after two cycles of transfections of cells with siRNA (Western blotting analysis). A and B, right panels, HEK293 and MLE-12 cells were exposed to TNFα (20 ng/ml), and mRNA levels were determined by RT-PCR at the time points indicated (n = 3). C, OGG1-dependent expression of CCL20 and CXCL1 mRNA. Cells were transfected with control siRNA (□) or siRNA to OGG1 (■) and TNFα-exposed (20 ng/ml). In controls, expression from B2M was tested. mRNA levels were determined at 30 min by RT-PCR (n = 3). D, depletion of NEIL1 and NEIL2 has no significant impact on TNF mRNA levels. Parallel cultures of cells were sequentially transfected with control siRNA (Cont) or siRNA to NEIL1, NEIL2, or OGG1. Inset, Western blotting analysis of protein levels. TNFα (20 ng/ml) was added for 30 min, and RNAs were isolated. mRNA levels of TNF were determined by RT-PCR (n = 3). E, IκB kinase inhibitor BMS-345541 decreases gene expression. Cells were pretreated with BMS-345541 (10 μm) for 3 h, and TNFα-exposed (20 ng/ml). mRNA levels were determined at 30 min by RT-PCR (n = 4). F, decreases in luciferase mRNA expression driven by TNF promoter in OGG1-depleted cells. OGG1 was depleted by siRNA, and cells were transfected with pGL4.2-Luc vector containing a promoter region (−974 + 90 bp) of human TNF (see under “Experimental Procedures”). mRNA expression levels of firefly luciferase were assessed at 30 min after mock (□) or TNFα (■) exposure (n = 4). G, NF-κB-dependent expression from the TNF promoter. Cells were co-transfected with IκB super-repressor (IκBSR) and pGL4.2-Luc, and then mock-exposed (□) or TNFα-exposed (■, 20 ng/ml). mRNA levels of firefly luciferase were assessed by RT-PCR (n = 3). ** = p < 0.01; *** = p < 0.001. HEK293 cells were utilized for experiments described in C–G. OGG1, 8-oxoguanine DNA glycosylase-1; CCL20, mRNA encoded by chemokine (C-C motif) ligand 20 gene; CXCL1, mRNA encoded by chemokine (CXC motif) ligand 1 gene; B2M, mRNA encoded by β2-microglobulin gene; IκBSR, super-repressor of κ-light-chain-enhancer of activated B cells; pGL4.2, luciferase (Luc) reporter vector 4.2; NEIL1, gene human Nei-like DNA glycosylase-1; NEIL2, gene human Nei-like DNA glycosylase2; OGG1, gene of human 8-oxoguanine DNA glycosylase-1; Tnf-Luc, human TNF promoter-containing pGL4.2-Luc expressing vector.