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Fig. 3. mDia1 mutants that affect MyoD expression cause G1 arrest without differentiation. (A) Cell cycle analysis of C2C12 myoblasts transfected with GFP-tagged ${Delta}$ N3 or N3HindIII 24 hours after transfection. Transfected (T) and untransfected (U) cells were distinguished by gating on GFP. (B) A greater proportion of ${Delta}$ N3 and ${Delta}$ N3HindIII transfected cells showed a G1 DNA content compared with untransfected cells (mean ± s.e.m., n=4, P<0.0002). (C,D) ${Delta}$ N3 transfected cells show reduced BrdU incorporation. Immunodetection of BrdU (green) in cells transfected with Flag-tagged mDia ${Delta}$ N3 (red) (mean ± s.d., n=3). (E,F) ${Delta}$ N3 transfected cells do not differentiate. ${Delta}$ N3 transfected cells were stained for Myogenin and p21, after 24 hours in differentiation medium (mean ± s.e.m., n=2). Arrows indicate transfected cells that are negative for two markers of differentiation: p21 (top) or Myogenin (middle), but positive for p27, a marker of reversible arrest (bottom). (G) Quantification of p27 induction: mDia mutants that affect MyoD expression ( ${Delta}$ N3, ${Delta}$ N3HindIII, H+P) induce expression of p27 (mean ± s.e.m., n=4).

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