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. 2015 Mar 12;112(13):3985–3990. doi: 10.1073/pnas.1503152112

Fig. 4.

Fig. 4.

STAT3 is dimethylated on K49 by EZH2. (A) The sequence of STAT3 surrounding K49 is aligned against the EZH2 target sequence surrounding H3 K27 and the SET9 target sequence surrounding H3 K4. Knockdown of EZH2 in A4 cells expressing WT-STAT3 was confirmed by Western analysis. To determine whether EZH2 knockdown impairs STAT3 Y705 phosphorylation, cells with or without EZH2 knockdown were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 1 h and were analyzed for Y705 phosphorylation. (B) EZH2-knockdown and control WT-STAT3 cells were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 4 h and were analyzed for K49 dimethylation, Y705 phosphorylation, and total STAT3. (C) WT-STAT3 and K49R-STAT3 cells were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL), and Y705 phosphorylation of STAT3 was determined. (D) WT-STAT3 cells, with or without EZH2 knockdown, were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 4 h, and the relative expression of the SERPINA1, SOCS3, and GADD45G genes was determined by qPCR. Values are shown as the means, with SDs, from triplicate experiments. (E) EZH2-knockdown and control hTERT-HME1 cells were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 4 h and were analyzed for EZH2 knockdown, K49 dimethylation, Y705 phosphorylation, and total STAT3 expression. (F) hTERT-HME1 cells, with or without EZH2 knockdown, were treated with IL-6 (50 ng/mL) and sIL-6R (62.5 ng/mL) for 4 h, and the relative expression of the SOCS3 and BCL3 genes was determined by qPCR. Values are shown as the means, with SDs, from triplicate experiments.