Figure 4.

miR-17-92 and ENH1 modulate Id1 subcellular distribution. (a) Western blot analysis shows that the Id1 protein is downregulated during C2C12 myoblast myogenesis. (b) Nuclear and cytoplasmic Id1 proteins are downregulated in C2C12 cells at DM4. Alpha-tubulin and TATA-binding protein (TBP) served as the loading controls. The data are presented as the means of the samples from three different cell samples. (c) Localization of exogenous Flag-Id1 in C2C12 myoblasts (cultured in GM) and in differentiating C2C12 cells at DM4, as detected by Flag-Id1 immunostaining (red). Nuclei were stained blue with DAPI. Scale bar, 50 μm. (d) si-ENH1 increases the nuclear Flag-Id1 in C2C12 myoblasts. C2C12 myoblasts were co-transfected with Flag-Id1 and si-ENH1 and transferred to DM for 4 days. The rest is as in c. (e) Western blot analysis of the whole-cell, nuclear and cytoplasmic Id1 proteins in C2C12 myoblasts transfected with si-ENH1 or NC. The rest is as in b. (f) Overexpression of miR-17, -20a or -92a induces the accumulation of Flag-Id1 in the nucleus. C2C12 myoblasts were co-transfected with Flag-Id1 and miRNA mimics and transferred to DM for 4 days. The rest is as in c. (g) Overexpression of miR-17, -20a or -92a regulates the protein levels of ENH1 and Id1 in C2C12 cells that were transfected with miRNA mimics or NC at GM and at DM4. Beta-actin served as the loading control. The rest is as in b. (h) Western blot analysis of nuclear and cytoplasmic Id1 in the C2C12 myoblasts transfected with a mixture of miR-17, -20a and -92a mimics or NC. The rest is as in b. (i) Id1 mRNA is upregulated in C2C12 myoblasts stably expressed miRNA as indicated. The data are presented as the means±S.D. of the samples from three different cell samples. *P<0.05 and **P<0.01