Fig. 3.

HDAC9 interacts with BRG1 and the lncRNA, MALAT1. a Increased expression of HDAC9 protein is noted in TGFR2^G357W or ACTA2^R179H expressing alleles by western blot when compared to other HDAC isoforms in human VSMCs. Three experimental replicates are quantified. b Immunoprecipitation of BRG1 demonstrates HDAC9 binding in TGFR2^G357W and ACTA2^R179H expressing mutant cells. Quantification of protein levels of three replicate experiments. c Top, CLIP-seq analysis of anti-HDAC9 and anti-BRG1 immune complexes illustrated by Venn diagram showing the number of transcripts detected by purification with both antibodies at baseline or during expression of TGFR2^G357W or ACTA2^R179H alleles. Bottom, cellular function of 107 core transcripts by ingenuity pathway analysis. d Quantitative PCR analysis of known cardiovascular lncRNAs during the expression of TGFR2^G357W or ACTA2^R179H alleles. e Quantitative PCR analysis of cardiovascular lncRNAs associated with either anti-HDAC9 or anti-BRG1 immune complexes. Four experimental replicates are quantified. f Serial immunoprecipitation of BRG1 and HDAC9 demonstrates the association of the lncRNA MALAT1 with BRG1-HDAC9 immunocomplexes. Quantification of protein levels of three replicate experiments. g Western blot analysis of HDAC9 and BRG1 proteins captured through UV crosslinking of bead immobilized biotinylated MALAT1 or LacZ antisense RNA probes. Three replicates are quantified. h Immunofluorescence of HDAC9 and BRG1 demonstrate lack of nuclear localization of HDAC9 in siMALAT1-treated cells. Bar = 15 µM, circles indicate nuclei location in siMALAT-treated cells, arrows indicate apparent cytoplasmic HDAC9 aggregates. i Semiquantitative western blot analysis of HDAC9 and HDAC1 from nuclear extracts (NE) and cytoplasmic extracts (CE) from control and siMALAT1-treated VSMCs. j siMALAT1 treatment of VSMCs decreases TGFR2^G357W or ACTA2^R179H mediated induction of MMP activity by in vitro gelatin zymography. Bar graphs are presented as mean with error bars (±S.D.), Student’s T-test, *p < 0.05, **p < 0.01 vs. WT. Full-length western blots presented in Supplementary Fig. 8