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. 2015 Aug 1;29(15):1605–1617. doi: 10.1101/gad.263574.115

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© 2015 Lee et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 3. — Knockdown of SMAD4 promotes myogenesis of old myoblasts. (A, top) Lysates of four independently isolated young and old myoblasts were subjected to immunoblot analysis with antibodies to detect the indicated proteins, and ACTB was used as a loading control. (Bottom) Quantification of Western blotting signals is presented as the means ± SD. (*) P < 0.05. (B, top) Twenty-four hours after old myoblasts were transfected with siCtrl and siSmad3 or siSmad4, myogenesis was induced using differentiation medium. Differentiation markers MyHC mRNA and Myog mRNA were identified by RT-qPCR analysis. (Bottom) The effect of SMAD4 knockdown by assessing the same parameters. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001. (C) Immunofluorescence staining for MyHC (green) and DAPI (blue) to detect differentiated myotubes in old myoblasts expressing normal or silenced levels of SMAD4. Bar, 200 µm. (Right) The fusion index was measured by the percentage of MyHC-positive cells that contained two or more nuclei versus total MyHC-positive cells. Six different views were randomly selected for measurement of the fusion index. (*) P < 0.05. (D) Expression levels of the indicated proteins, as determined by Western blot analysis in young and old myoblasts cultured in differentiation medium for up to 3 d (DM3). ACTB was used as a loading control. (E) Relative expression of miR-431 in the cells described in D. The data were normalized to the amount of U6 snoRNA and were expressed relative to the normalized value for young myoblasts in growth medium. (GM) Growth medium; (DM) differentiation medium. (F) The effect of miR-431 on the repression of myogenesis due to TGF-β treatment. After transfection with M-Ctrl or M-miR-431, old myoblasts were incubated in differentiation medium with 5 ng/mL TGF-β1 for 2 d. The levels of MyHC mRNA and Myog mRNA were measured by RT-qPCR analysis. The results were normalized to Actb mRNA levels in each sample, as measured by RT-qPCR analysis. Data are presented as the means ± SD. (**) P < 0.01. (G) The effect of miR-431 on the SMAD signaling induced by the treatment with TGF-β1. C2C12 cells were transfected with M-miR-431 or I-miR-431 together with the 4xSBE-luc reporter construct. Cells were treated with 5 ng/mL TGF-β1 for 16 h before luciferase analysis. The values represent fold induction relative to the basal activity of 4xSBE-luc in a dual-luciferase assay. (*) P < 0.05.