HDAC1/2 regulated VCAM1 transcription through a mechanism involving transcription factor GATA6. (A) HAEC were pretreated with Romi (40 nM) at indicated doses for 1 h and co-cultured with 0.1 ng/mL TNFα for 2 h prior to quantitative PCR to measure VCAM1 and ICAM1 mRNA (n = 4-5). (B) After treatment with Romi or transfection with siRNA, HAEC were stimulated with TNFα for 4 h prior to Western blot analysis (n = 3). Shown below is quantification of GATA6 protein. (C) HAEC were transfected with luciferase reporter plasmid of GATA, treated as in (A) followed by dual-luciferase assay to detect the transcriptional activity of GATA (n = 4). (D) HAEC were treated as in (A), then steady-state GATA6 mRNA was quantified (n = 4-5). (E) After sequential treatment with Romi (40 nM) and stimulation with TNFα (0.1 ng/mL), heterogeneous nuclear GATA6 RNA (GATA6 hnRNA) (n = 4) was analyzed. (F) Activity of pGL3 firefly luciferase (luc) construct containing wild type VCAM1 promoter or GATA-binding site mutation (VCAM1 Mut) was measured with dual-luciferase reporter system (n = 5). *p < 0.05; **p < 0.01; ***p < 0.001 vs ctrl repeated measures one-way ANOVA followed by Dunnett's test (A, D) or by Tukey's test (B, F); one sample t-test (C, E).