Figure 3

FL- Irs-1 transcript reduces Rb mRNA levels. (A) Expression levels of Rb mRNA in C2C12 myoblasts (GM) and myotubes (DM4). (B) Partial sequence complementarity between the 5′UTR of FL-Irs-1 transcript (nt 373 – nt 405) and Rb mRNA (nt 131 – nt 165). (C) Schematic representation of position of complementary element in the 5′UTR of FL-Irs-1 transcript. A position of an LNA-based antisense oligonucleotide (FL-5′-AS-ODN) is shown. (D) Effect of FL-Irs-1 5′UTR on Rb mRNA expression levels. C2C12 myoblasts were untransfected (−) or transfected with an Rb mRNA-expressing plasmid in combination with the indicated effector plasmid (pFL-5′UTR, pFL-5′UTR-del). Rb mRNA levels were measured by qRT-PCR. Mean ± SD, n = 4-5, *p < 0.01 versus control transfectant (pRB + pLuc). (E) Effect of FL-5′-AS-ODN on Rb mRNA reduction by FL-Irs-1 5′UTR. C2C12 myoblasts were co-transfected with plasmids expressing Rb mRNA and the FL-Irs-1 5′UTR RNA in combination with the indicated AS-ODN. Mean ± SD, n = 4-5, *p < 0.01 versus control transfectant (pRB + pLuc). (F) Effect of knockdown of endogenous FL-Irs-1 transcript on endogenous Rb mRNA levels. C2C12 myoblasts were transfected with the indicated shRNA or siRNA. Mean ± SD, n = 4, *p < 0.01 versus no treatment (none). (G) Effect of supplementary expression of IRS-1 protein on Rb mRNA levels under FL-Irs-1 transcript knockdown conditions. C2C12 myoblasts were transfected with the sh-FL-Irs-1 plasmid in combination with a plasmid encoding IRS-1 protein fused to V5 tag (pIRS-1-V5). Expression levels of exogenous IRS-1 protein were determined by immunoblot analysis using anti-V5 antibody. Mean ± SD, n = 3, *p < 0.01 versus control transfectant (sh-Control).