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Figure 3


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).