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

siRNAs were designed to target regulator genes in modules dark-turquoise and orange, and transfected into murine C2C12 muscle cells in vitro. Relative mRNA expression was measured by qPCR 48 hours after transfection. Expression was normalized to Hrrt1 and Ppia internal controls. a) Zfp36l2 as a partial regulator of genes in module dark-turquoise. Expression of Zfp36l2 was significantly reduced relative to its expression in control cells at 48 hours post-transfection of siRNA. Expression of other genes (Fam134b, Irs2, Ndel1, Nr4a3, Ppargc1a, Crem, Sdc4) in module dark-turquoise was significantly reduced; these genes are functionally enriched in apoptosis and cell death. b) Cxcr7 as a regulator of genes in module orange. siRNA targeting significantly reduced the levels of Cxcr7 relative to control at 48 hours post-transfection. Expression of most top hub genes in module orange, like Egr1, Zfp36, Fos, Klf4, Ankrd1, Otud1, Adamts1, Gadd45b, Ier5, Tiparp, and Jun, 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.