Fig. 2

EZH2 is a potential downstream target of STAT3 signaling. a EZH2 mRNA expression was decreased in SGC7901 cells transfected with siSTAT3. b The protein level of EZH2 expression was downregulated in SGC7901 cells using knockdown of STAT3 with siRNA. c The expression of STAT3 status and EZH2 was induced by IL-6 in SGC7901 cells. d Luciferase activity was measured in extracts from SGC7901 cells transfected with different luciferase reporter constructs, containing the full-length promoter (Region 1) or the region only containing STAT3-binding sites (Region 3) or not (Region 2); luciferase activity normalized for Renilla luciferase activity and expressed relative to the activity of the untreated group; the higher activity of EZH2 was detected in Region 1 and Region 3, which contained STAT3-binding sites (Fig. 2d, P < 0.001). e Dual luciferase reporter analysis of EZH2 promoter. The construct with full-length of EZH2 promoter (-1702/+52) was inactivated by siSTAT3 treatment with or without IL-6 stimulation. f The specific region (−436/+48) of EZH2 promoter was detected by ChIP-PCR. STAT3 mediated fold-enrichment of STAT3-binding regions of EZH2 promoter. Further, the binding activity was increased by IL-6 stimulation compared with the untreated group (P = 0.0059). When knockdown of STAT3 using siRNA, the binding activity was decreased with or without IL-6 (P = 0.0043). g The nuclear extracts from SGC7901 cells were incubated with biotin-labeled wild-type before the mutant or cold probes were added for 20 min. The specific region of the STAT3 stand motif (−222/−197) demonstrated effective binding ability. All the results for EZH2 activity were analyzed by EMSA. The results in (d) and (e) are represented as mean ± SD values