Fig. 6. mDia negatively regulates TCF. (A) TCF activity is suppressed by mDia1N3 and activated by mDia knockdown. (i) Myoblasts were co-transfected with the TCF reporter plasmid TOP-flash + GFP (control) or mDia1N3 (N3) and TCF-dependent luciferase activity measured. Values represent normalized ratios of TOP-flash activity to the respective FOP-flash control (mean ± s.e.m., n=11, P<0.0001). (ii) Myoblasts were co-transfected with TOP-flash + mU6 vector (control) or mDia1 shRNA (shRNA) and luciferase activity measured as in (i) (mean ± s.e.m., n=5, P<0.0021). (iii) Comparison of effects of full-length (FL) and different mDia mutants on TCF activity (values represent normalized TCF activity, mean ± s.e.m., n=2). N3 is the most effective at suppressing TCF activity. (B) Inhibition of TCF suppresses MyoD expression. Cells were transfected with GFP alone (control) or along with dominant negative TCF-1E (DN TCF-lacking the -catenin-binding domain) and MyoD expression quantified (mean ± s.e.m., n=3, P<0.0021). (C) SRF activity is not affected by -catenin S37A and dnTCF. Myoblasts were co-transfected with the SRF reporter with control (pBS), N3, S37A or DN TCF constructs and luciferase activity measured. (mean ± s.e.m., n=4).