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. 2016 Feb 4;7(2):e2086. doi: 10.1038/cddis.2016.10

Figure 5.

Figure 5

MiR-222 overexpression and Rbm24 silencing inhibit muscle alternative splicing during early myoblasts differentiation. (a) MSC myoblasts transfected in GM with siGFP (siGFP), miR-222 (222) or siRbm24 (siRbm) duplex RNAs were shifted to DM for 8 h, 16 h or 24 h and analyzed for expression of Rbm24, p27 and Myogenin proteins by western blot. The table shows a quantification of the expression of Rbm24, p27 and Myogenin proteins normalized to p38, relative to control siGFP, referred as 1. Untransfected MSC in GM (GMnt) and DM (DMnt) at 24 h are shown for comparison. (b) RNA from parallel cell cultures was analyzed by semi-quantitative RT-PCR and amplicons were separated on ethidium bromide-stained agarose gels to determine splicing efficiency of muscular isoforms of Coro6 and Fxr1 transcripts. Greyscale of images was inverted for a sharper band definition. In the scheme, the black rectangles represent muscle-specific exons and the black arrows indicate primer positions. (c) General and muscular isoforms of NACA transcripts were detected by qPCR analysis and normalized to GAPDH transcript. Expression of muscle-specific isoform of NACA (skNAC) over the general NACA isoform, and relative to siGFP, referred as 1, is shown in the histogram. A representative experiment is shown