Fig. 4

STAT1 mediates E2F2 regulation of interleukin (IL)-1α, IL-1β, and tumor necrosis factor (TNF)-α expression. a–d Effect of E2F2 on STAT1. E2F2 was overexpressed by adenovirus infection or inhibited by small interfering RNA (siRNA) with or without lipopolysaccharide (LPS; 10 μg/mL). qRT-PCR (a,b) and Western blot (c,d) were performed to detect expression of STAT1. e Schematic representation of STAT1 promoters, primers for the ChIP assay, and the E2F2 binding motif in the STAT1 promoter. ChIP (f) and luciferase (Luc) reporter assays (g) were performed to show that E2F2 was recruited to the STAT1 gene promoter in RASFs in the presence of LPS. Nuclear and cytoplasmic proteins were fractionally extracted from E2F2 knocked-down RASFs (h) and E2f2^−/− MEFs (i). Effects of E2F2 on nuclear translocation of STAT1 were determined by Western blot. (Lamin A/C as a reference for nuclear extraction (N); Tubulin as a reference for cytoplasmic extraction (C).) Effect of E2F2 on nuclear translocation of STAT1 was observed using confocal fluorescence microscopy both in E2F2-silenced RASFs (j) and E2f2^−/− MEFs (k). STAT1 (green) was detected using anti-STAT1 antibody. Nuclei were stained with DAPI (blue). l In E2F2-overexpressing RASFs, IL-1α, IL-1β, and TNF-α were analyzed by qRT-PCR after silencing STAT1 in the presence of LPS stimulation (10 μg/mL). m In STAT1-overexpressing RASFs, IL-1α, IL-1β, and TNF-α were analyzed by qRT-PCR after silencing E2F2 in the presence of LPS stimulation (10 μg/mL). n In E2f2^−/− MEFs, expression of IL-1α, IL-1β, and TNF-α was detected using qRT-PCR after STAT1 overexpression in the presence of LPS stimulation (10 μg/mL). The results shown are means ± SEM of three independent experiments performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001, versus the control. Ad-GFP adenovirus encoding green fluorescent protein, NC knockdown scramble control, ns not significant, siE2F2 small interfering RNA knockdown of E2F2, WT wild-type