Figure 4.

Blocking STAT3 acetylation at Lys685 disrupted DNMT1-STAT3 interaction and reversed the suppressive effects of HDAC1/2 inhibition on GATA6 and VCAM-1 expression in HAEC. (A) HAEC were pretreated with Niclosamide (2 μM, Niclo) for 1 h, stimulated with TNFα for 4 h followed by Western blotting (n = 4). (B-C) After transfection with empty vector (sham), vector containing wild type STAT3-coding sequence (WT), or sequence bearing K685R mutation (K685R), HAEC were pretreated with Niclo (C) or Romi (D) for 1 h and stimulated with TNFα for 4 h prior to Western blot analysis (n = 3). For A-C, shown below is quantification of VCAM-1, GATA6 and Ac-STAT3 protein. (D) After overexpression with WT STAT3 or K685R mutant, HAEC were treated as above followed by MSP assay to evaluate GATA6 methylation at +144/+255 with agarose gel electrophoresis and real-time PCR (n = 4) that was carried out with methylated cytosines-targeting primers and the data were normalized to UBB expression. (E) HAEC were pretreated with Romi and stimulated with TNFα, followed by immunoprecipitation with anti-DNMT1 or anti-STAT3 antibody (n = 3). (F) After treatment, enrichment of DNMT1 onto +140/+255 of GATA6 promoter was evaluated with chromatin immunoprecipitation assay (n = 3). (G) HAEC were transfected with WT STAT3 or K685R mutant, pretreated with Romi and stimulated with TNFα, followed by immunoprecipitation as above (n = 3). Shown are representative images. The ratio between STAT3 and DNMT1 was quantified and shown below. *p < 0.05, **p < 0.01, **p < 0.001, repeated measures one-way ANOVA followed by Tukey's test (A, B, C, D, F); one sample t-test (G).