Knockdown of regulator genes by RNA interference reveals regulation within modules.

<p>siRNAs were designed to target regulator genes in modules dark-turquoise and orange, and transfected into murine C2C12 muscle cells <i>in vitro</i>. Relative mRNA expression was measured by qPCR 48 hours after transfection. Expression was normalized to <i>Hrrt1</i> and <i>Ppia</i> internal controls. a) <i>Zfp36l2</i> as a partial regulator of genes in module dark-turquoise. Expression of <i>Zfp36l2</i> was significantly reduced relative to its expression in control cells at 48 hours post-transfection of siRNA. Expression of other genes (<i>Fam134b</i>, <i>Irs2</i>, <i>Ndel1</i>, <i>Nr4a3</i>, <i>Ppargc1a</i>, <i>Crem</i>, <i>Sdc4</i>) in module dark-turquoise was significantly reduced; these genes are functionally enriched in apoptosis and cell death. b) <i>Cxcr7</i> as a regulator of genes in module orange. siRNA targeting significantly reduced the levels of <i>Cxcr7</i> relative to control at 48 hours post-transfection. Expression of most top hub genes in module orange, like <i>Egr1</i>, <i>Zfp36</i>, <i>Fos</i>, <i>Klf4</i>, <i>Ankrd1</i>, <i>Otud1</i>, <i>Adamts1</i>, <i>Gadd45b</i>, <i>Ier5</i>, <i>Tiparp</i>, and <i>Jun</i>, was also reduced. The data represent mean±SEM (n = 4–6 independent experiments). * indicated significant level at p<0.05 and ** indicated significant level at p<0.01.