Chemical inhibition or RNAi knockdown of HDAC3 in human SVG-A cells suppresses expansions.

<p>(A) The genetic assay is essentially as described <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001257#pbio.1001257-Claassen1" target="_blank">[27]</a>. Cells were treated with either HDAC inhibitor <b>4b</b>, compound <b>3</b>, or DMSO only. Alternatively, siRNA was used with scrambled siRNA as a control. Expansions are scored using yeast as a biosensor, and total plasmid counts are monitored by bacterial transformation for enhanced sensitivity. (B) Expansion frequencies as a function of inhibitor dose, compared to DMSO-treated control cells. Blue, <b>4b</b>-treated; red, compound <b>3</b>-treated. Error bar, ±SEM; * <i>p</i><0.05 compared to DMSO-treated cells. Details in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001257#pbio.1001257.s011" target="_blank">Table S3</a>. (C) Expansion frequency after RNAi. Knockdown efficiency, judged by three independent immunoblots, averaged 76(±8)% for HDAC3 and 76(±2)% for HDAC1. Error bars, ±SEM; * <i>p</i><0.05 compared to scrambled control. Details in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001257#pbio.1001257.s011" target="_blank">Table S3</a>. (D) Expansion sizes, derived from PCR analysis. 21 genetically independent expansions for DMSO, 16 for <b>4b</b> (combined data from 10 µM and 20 µM treatments), 28 for scrambled siRNA, and 13 for HDAC3 siRNA. (E) Cell viability measured by nigrosin staining just prior to cell harvest. (F) Representative immunoblot of acetylated histone H4 and total histone H4 upon treatment with <b>4b</b>; data summary in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001257#pbio.1001257.s006" target="_blank">Figure S6</a>. (G) Expansion frequencies after RNAi against histone acetyltransferases. Error bars, ±SEM; * <i>p</i><0.05 compared to scrambled control.