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. 2008 Aug 11;182(3):497–507. doi: 10.1083/jcb.200801119

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© 2008 Kang et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 8. — RNAi-mediated depletion of Cdc42GAP enhances myogenic differentiation. (A) Lysates of C2C12 cells stably transfected with pSilencer containing one of three independent Cdc42GAP siRNA sequences (designated 1, 2, and 3) or pSilencer containing an irrelevant sequence (−) were Western blotted with Cdc42GAP or, as a control, pan-cadherin antibodies. (B) Levels of GTP-bound and total Cdc42 in C2C12 cells that express Cdc42GAP siRNA (+) or an irrelevant sequence (−) in DM were assessed as described in Fig. 6 A. (C) Photomicrographs of C2C12 cells that express Cdc42GAP siRNA sequences or an irrelevant (pSilencer) sequence as indicated, cultured in DM, fixed, and stained with an antibody to MHC. Bar, 0.1 mm. (D) Quantification of myotube formation. Values represent means of triplicate determinations ±1 SD. The experiment was repeated three times with similar results. Asterisks indicate difference from pSilencer control, P < 0.02. A level of myotube formation by control cells (∼45% nuclei in MHC⁺ cells) was selected so as to permit visualization of enhanced differentiation by Cdc42GAP siRNA. (E) Western blot analysis of MHC expression by C2C12 cells that express Cdc42GAP siRNA sequences numbers 1 or 3 or an irrelevant sequence (−), cultured in DM for 48 h.