A dominant negative form of RhoA blocks differentiation of P19CL6 cells into a cardiomyocyte-like phenotype. P19CL6-derived cell lines were grown in growth medium or differentiation medium (growth medium containing 1% DMSO) in 24-well culture dishes from day of seeding and induction (D0) for 16 days (D16). Differentiated cardiomyocyte clusters (D16) were detected using a cardiac troponin-I (cTnI) antibody. (a)–(f) P19CL6 cell lines processed with cTnI antibody: wt P19CL6, wild-type (nontransfected) P19CL6 cells; P19CL6 mock, P19CL6 cells stably transfected with expression plasmid alone; P19CL6mRhoAN19, P19CL6 cells stably transfected with plasmid expressing dominant negative mouse RhoAN19; (g) primary antibody (Ab) control (i.e., lacking primary antibody); (h) nonimmune mouse serum (NIS) control. Addition of 1% DMSO resulted in differentiation into a cardiomyocyte-like phenotype in P19CL6 cells and the mock transfected P19CL6 cell lines but not in the dominant negative mutant (P19CL6mRhoAN19) cells. (b), (d) Beating clusters, once processed for cTnI immunodetection displayed cTnI-positive cell clusters (red/brown staining). Bar = 10 µM.
