Overexpression of HIVEP1 inhibits NF-κB activity upon Toll-like receptor stimulation and binds to the TNF and TNFAIP3 promoter regions. (A) HIVEP1 protein expression in HEK293T cells transfected with HIVEP1 expression vector (1 µg) or control plasmid (B–D) CD14/TLR2 HEK293T cells were transfected with 83 ng of HIVEP1 or control vector together with 33 ng of NF-kB and 0.7 ng of Renilla constructs and incubated with medium or PAM3CSK4 (1 µg/mL); overexpression of HIVEP1 was associated with reduced activity of NF-κB (B), and reduced expression of TNF-α mRNA (C) and A20 mRNA (D). HEK293T cells transfected with MyD88 (E) or TRAF6 (F) (or control cells) were co-transfected with increasing doses of HIVEP1 expression vector (0, 30 or 150ng); these MyD88 or TRAF6 transfected cells were not stimulated with PAM3CSK4; overexpression of HIVEP1 was associated with a dose-dependent inhibition of MyD88 or TRAF6 driven NF-κB activity. mRNA expression was normalized to HPRT mRNA. Data shows means ± SEM. (G, H) HEK293T cells were transfected with 30 µg of HIVEP1 expression vector or control plasmid. 24 hours after transfection, the cells were collected for CHIP analysis. RT-qPCR amplification was performed with total DNA and immunoprecipitated DNA using TNF and TNFAIP3 promoter primers spanning the NF-κB binding regions. Data shows means ± SEM and presented as a percentage of input DNA. Data shown were pooled from two (A,B,C), three (E, F) independent data sets (four replicates samples for each condition within each experiment) and three (G, H) independent data sets (two duplicate samples for each experiment)*P < 0.05, **P < 0.01, ***P < 0.001.
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