Fig. 5

MyD88 mediates regulation of the PI3K/AKT/NF-κB pathway by E2F2. a,b E2F2 can regulate expression of PI3K/AKT/NF-κB. E2F2-silenced RASFs and E2f2^−/− MEFs were cultured with or without lipopolysaccharide (LPS). Western blot was performed to detect phosphorylation of AKT and NF-κB P65 both in E2F2-silenced RASFs (a) and E2f2^−/− MEFs (b). c,d Effect of E2F2 on translocation of p65. E2F2 knocked-down RASFs (c) and E2f2^−/− MEFs (d) were cultured under LPS stimulation (10 μg/mL) for 12 h; nuclear and cytoplasmic proteins were extracted separately and then Western blot was performed (Lamin A/C as a reference for nuclear extraction (N); Tubulin as a reference for cytoplasmic extraction (C).) e,f Effects of E2F2 on p65 nuclear translocation both in RASFs (Fig. 4e) and MEFs (Fig. 4f) observed using confocal fluorescence microscopy. g–j E2F2-silenced RASFs and E2f2^−/− MEFs were cultured with or without LPS. qRT-PCR (g,h) and Western blot (i,j) were performed to detect the effect of E2F2 on MyD88. k Schematic representation of the MyD88 promoter, primers for the ChIP assay, and the E2F2 binding motif in the MyD88 promoter. l,m E2F2 was recruited to the MyD88 gene promoter in RASFs in the presence of LPS. ChIP (l) and luciferase (Luc) reporter assay (m) were performed in RASFs and MEFs in the presence or absence of LPS (10 μg/mL). n MyD88 mediated the effect of E2F2 on PI3K/AKT/NF-κB pathways. Western blot showed that knockdown of MyD88 could significantly inhibit the phosphorylation of AKT and P65 in the presence or absence of E2F2 overexpression. qRT-PCR showed that inhibition of MyD88 can inhibit the expression of inflammatory factors and significantly reduce the upregulation of interleukin (IL)-1α (o), IL-1β (p), and tumor necrosis factor (TNF)-α (q) by E2F2. The effect of inhibitors of PI3K/AKT/NF-κB pathways (LY294002 and PDTC) on E2F2; qRT-PCR was used to detect the effect of inhibitors on expression of IL-1α (r,u), IL-1β (s,v), and TNF-α (t,w) in response to LPS. The results shown are means ± SEM of three independent experiments performed in triplicate. **P < 0.01, ***P < 0.001, versus the control. Ad-GFP adenovirus encoding green fluorescent protein, NC knockdown scramble control, si small interfering, WT wild-type