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. 2012 Dec 15;23(24):4778–4785. doi: 10.1091/mbc.E12-04-0311

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© 2012 Ling et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — Sharp-1 inhibits differentiation with increased H3K9me2. (A) Sharp-1 expression in pBabe (control) and pBabe-Sharp-1 C2C12 cells was analyzed by Western blot. (B–D) Differentiation of control and pBabe-Sharp-1 cells was analyzed with anti-MHC antibody. Nuclei were stained with DAPI (B). The myogenic index was determined and is represented as mean ± SD (C). Lysates from undifferentiated myoblasts (day 0) and after differentiation (days 1 and 3) were examined for myogenin and troponin T (D). (E, F) ChIP assays were performed at days 0 and 2 on the myogenin promoter, using H3K9me2 and H3K9K14ac antibodies. (G) C2C12 cells were transfected with scrambled siRNA or Sharp-1 siRNA (siSharp-1). The down-regulation of endogenous Sharp-1 was analyzed by Western blot. (H–J) Differentiation in siRNA and siSharp-1 cells was quantified by immunofluorescence analysis of MHC⁺ myotubes (H), myogenic index (I), and expression of myogenin and troponin T (J). (K) H3K9me2 enrichment was analyzed by ChIP in siRNA and siSharp-1 cells in undifferentiated (day 0) and differentiated cells (day 2).