Fig. 5
HIF-1α and STAT3 cooperatively regulate Il10 transcription. a Quantitative RT-PCR analysis of Il10 mRNA expression in WT splenic B cells cultured under normoxia or hypoxia for indicated time (n = 5 per group). b Scheme of Il10 promoter indicating the predicted HRE regions (I, II, III, IV, and V). Enrichment of HIF-1α (c) and H3K4me3 (d) in HRE regions of Il10 promoter in B cells under normoxia or hypoxia for 24 h (n = 3 per group). e Luciferase activity in 293T cells transfected with pGL3 empty vector (EV), HRE constructs (I, II, III, IV, and V) under normoxic or hypoxic conditions for 24 h (n = 3 per group). The Luc/β-gal ratio was normalized to EV at 20% O2. f ChIP assays in enriched splenic B cells showing the recruitment of the endogenous HIF-1α on the HRE regions of Il10 promoter after anti-IgM stimulation for 8 h (n = 3 per group). g Co-immunoprecipitation of HIF-1α and pSTAT3727 in enriched splenic B cells stimulated with anti-IgM for 8 h. The whole cell lysates were immunoprecipitated with either anti-HIF-1α or anti-pSTAT3727 antibodies. h ChIP on ChIP assays in enriched splenic B cells pulling down sequentially HIF-1α and pSTAT3727 showing the HIF-1α-pSTAT3727 binding on the HRE regions of Il10 promoter after stimulation with anti-IgM for 8 h (n = 3 per group). Data are shown as mean ± s.e.m. Pictures are representative of four (a) or three (c–h) independent experiments. **P < 0.01 and ***P < 0.001 (two-tailed unpaired Student’s t-test) (see also Supplementary Figure 5)
