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Fig. 7. Transfection of wild-type or dominant-interfering RhoA, or C3 transferase leads to arrest. Asynchronous myoblasts were co-transfected with plasmids encoding nlsGFP alone (GFP), WT RhoHA alone (RhoA/WT Rho), GFP + RhoAN19 (DN Rho) or GFP + C3 transferase (C3 Tr). After 24 hours, cells were pulsed with BrdU. (A) Co-detection of transfection marker and BrdU. WTRhoHA transfectants were detected using an anti-HA tag antibody (red); co-transfected nlsGFP was used to detect DN Rho transfectants (green). Anti-BrdU was detected using streptavidin-Alexa Fluor 350 (blue). Arrows indicate BrdU-negative untransfected cells (top) and transfected cells (middle and bottom). Scale bar: 10 µm. (B) Compared to control transfected cells, expression of WT Rho, DN Rho or C3 Tr led to suppression of DNA synthesis (mean ± s.d., n=3; ^*P<0.004, ^**P<0.001). (C) Wild-type RhoA and dominant-negative RhoA have divergent effects on MRFs. Asynchronously growing myoblasts were co-transfected as in A. Endogenous regulators were detected using immunofluorescence. Expression of WT Rho did not significantly affect MyoD expression, but increased the frequency of cells expressing myogenin and p21. By contrast, expression of DN Rho led to suppression of MyoD, and no induction of myogenin or p21 (mean ± s.d. n=3; ^*P<0.004, ^**P<0.001).

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