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. 2014 Apr 4;289(21):14633–14643. doi: 10.1074/jbc.M113.509158

FIGURE 3.

FIGURE 3.

VentX suppresses IL6 expression. A, monocytes were transfected with VentX MO or control MO and GM-CSF and IL4 were added into culture medium for 5 days. Cells were harvested for quantitative analysis of mRNA level of indicated genes. B, monocyte was transfected with pcDNA-GFP or pcDNA-GFP.VentX through electroporation. Cells were then cultured in the presence of GM-CSF and IL4 for 3 days, and GFP-positive cells were sorted out to analyze IL6 mRNA level. C, monocytes were transfected and treated as above, and cells were harvested for intracellular staining followed by flow cytometry analysis of IL6 levels. Results shown are representative of at least three independent experiments. D, −592 bp human IL6 promoter reporter was transfected with pcDNA-GFP or pcDNA-VentX plasmid into primary monocytes through electroporation. Two days after transfection, cells were harvested to analyze the luciferase activity. E, various IL6 promoter reporters were transfected into U2OS cells that express VentX upon induction with doxycycline. Cells were harvested 2 days after DOX addition, and luciferase activity in the presence or absence of DOX was analyzed. F, −80 bp IL6 promoter reporter with mutant NFκB binding site was transfected into U2OS cells. Then DOX was added into medium or omitted, and luciferase activity was determined 2 days after transfection. G, THP1 cells expressing GFP or GFP.VentX under the control of tetracycline-inducible promoter were treated with DOX for 2 days and harvested for ChIP assay with anti-NFκB/p65 antibody. The human IL6 promoter region containing an NFκB binding site was amplified by real-time PCR as described under “Experimental Procedures.” H, THP1 cells expressing GFP.VentX were transfected with control siRNA or IκBα siRNA, and cells were harvested 2 days after transfection for ChIP assay with anti-NFκB/p65 antibody as described above. Results shown are mean ± S.D. of triplicates from one representative of three independent experiments. I, −592 bp human IL6 promoter reporter was transfected with pcDNA-VentX plasmid into U2OS cells that have been transfected with control siRNA or IκBα siRNA 12 h prior. Two days after transfection, cells were harvested, and luciferase activity was determined. Error bars indicate standard deviation of representative experiments carried out in triplicate.