Trichostatin A and SMC differentiation markers.

<p><b>A</b>. Western blots of A404 cell histone extract, using anti-acetyl histone H4 or anti-histone H4 primary antibody. Cntrl  =  Control; RA  =  retinoic acid; TSA/RA  =  trichostatin A+ retinoic acid. Time 0 and 48 h of treatment are shown. <b>B</b>. Increased relative expression of SMC differentiation markers after 48 h in TSA + RA-treated A404 cells vs. RA-treated alone. For all ratios, p<0.05. Acta2  =  SM α-actin, Tagln  =  transgelin/SM22α, Cald1  =  caldesmon 1.</p

p300 interactome transcription factors.

<p>Transcription factors in the p300 interactome which demonstrated significant regulation during A404 differentiation vs. untreated cells (FDR<1). White  =  unchanged. Light grey  =  upregulated. Dark grey  =  downregulated. Gene number and % of interactome shown for each section.</p

p300 protein levels with SMC differentiation/serum-starvation.

<p>Plots of scanned, digitized Western blots probed with anti-p300, and representative blots. <b>A</b>. Top: A404 cells treated with retinoic acid for up to 96 hours. Bottom: A404 p300 Western blot. Two lanes per time point, same order as plot. <b>B</b>. Top: primary human coronary artery SMCs, serum starved for up to 72 hours. Bottom: CASMC p300 Western blot. Two lanes per time point, same order as plot. Integrated band densities were normalized to Gapdh and background. Mean ± standard deviation of lane duplicates are shown in density units.</p

p300 siRNA knockdown and human SMC differentiation markers.

<p><b>A</b>. Human CASMC were transfected with either scrambled negative control siRNA (siNeg) or with a transcript targeting EP300 (siEP300), and then either serum fed (Fed) or serum starved for 48 hours (SS48). Resulting qRT-PCR expression values were normalized to control (Fed cells transfected with siNeg). *  =  significantly (p<0.05) different from control. **  =  significantly (p<0.05) different from control and from siNeg time-matched controls. <b>B</b>. A404 cells were transfected with either scrambled negative control siRNA (siNeg) or with a transcript targeting Ep300 (siEp300), and then treated with retinoic acid for 24 (RA 24 h) or 48 hours (RA 48 h), or left untreated (No RA). Resulting qRT-PCR expression values were normalized to control (No RA cells, transfected with siNeg). *  =  significantly (p<0.05) different from control. **  =  significantly (p<0.05) different from control and from siNeg time-matched controls.</p

HDAC expression with SMC differentiation.

<p>Fold changes vs. control for histone deacetylase genes and SMC marker genes during A404 differentiation time-course. NS: not significant. For significant changes, FDR<1.</p

Heatmap and regulated chromatin remodeling genes.

<p><b>Left</b>: Row-normalized gene expression heatmap of A404 cells treated with all-trans retinoic acid for 96 hours, then puromycin for 48 hours, significant at FDR<1. Shown are 2,739 genes upregulated (P>C), and 2,227 genes downregulated (C>P). C = Control replicates, n = 6. P = Puromycin group replicates, n = 6. Green: down. Red: up. One gene/row. <b>Right</b>: Regulated chromatin remodeling genes during A404 differentiation. GO annotation terms for selected pairwise SAM comparisons (FDR<1) of treatment groups were obtained, using gene lists with unique names. Values are % of total set of 171 chromatin remodeling genes on array. Downregulated and Upregulated  =  vs. control A404 cells. RA48 and RA96  =  RA-treated for 48 or 96 h. Puro  =  RA-treated 96 h, then puromycin-treated for 48 h.</p

p300 HAT inhibition and trichostatin A.

<p>Area-under-the-curve (AUC) pixel densitometry was calculated for scanned, digitized anti-acetyl-histone H4 Western blots (shown at bottom) of A404 cells either untreated (Cntrl), or treated with trichostatin A+ retinoic acid (RA/TSA) or trichostatin A+ retinoic acid + Lys-CoA-TAT (LysCoA/RA/TSA) for 24 hours. Values shown are a ratio of anti-acetyl-histone H4:anti-histone H4, normalized to untreated control AUC ± standard error for three experiments. Western lanes (in duplicate) correspond to graph columns.</p

Gene regulation in human coronary SMCs by qRT-PCR.

<p>Cells were serum starved in basal medium for up to 72 h to induce differentiation. P300  =  EP300 HAT, ACTA2  =  SM α-actin, MYH11  =  SM-MHC. 48 and 72 h time points are shown as fold change from baseline ± standard error. All values are significantly increased vs. control (p<0.05).</p

Differential regulation of selected chromatin remodeling gene classes with SMC differentiation.

<p><b>A</b>. DNA methyltransferases. <b>B</b>. Histone methyltransferases. <b>C</b>. Histone acetyltransferases. <b>D</b>. SWI/SNF family. Row-normalized heatmaps are shown labeled with gene symbols. Control  =  untreated A404 cells. RA48/RA96  =  Retinoic acid treated for 48 and 96 h respectively. Puro  =  Treated with RA for 96 hours, then puromycin for 48 hours. Green: down. Red: up.</p

p300 HAT inhibition controls.

<p>A404 cells were treated for 24 hours with: 1) retinoic acid (RA), 2) 20 µM Lys-CoA-TAT (LysCoAT), or 3) 20 µM control TAT peptide (TAT). Expression levels obtained by qRT-PCR for SMC markers are shown as fold change from baseline untreated cells ± standard error. *  =  significantly (p<0.05) different from control.</p