RhoA and ERK1 inhibition abrogates the prohypertrophic effects of Dysbindin. (A) Expression of hypertrophic gene markers Nppa and Nppb was determined by qRT-PCR in Dysbindin-overexpressing NRVCMs in the absence or presence of C3 transferase, a RhoA inhibitor. LacZ-overexpressing cells were used as a control group. n = 6. (B) Representative images showing the phenotypic effect of C3 transferase. NRVCMs were cultured on coverslips in triplicates, infected with Adenovirus expressing Dysbindin/LacZ for 72 h, and immunostained with α-actinin. Nuclei were stained with DAPI. Cells were treated with C3 transferase 12 h before immunostaining. (C) Cell surface area of C3 transferase treatment datasets was measured from randomly selected 300 or more cells from three different coverslips using ImageJ software. (D) Expression of Nppa and Nppb was determined in Ad-LacZ– or Ad-Dysbindin–infected NRVCMs in the absence or presence of MEK1 inhibitor by qRT-PCR. n = 6. (E) Representative images showing the phenotypic effect of MEK1 inhibitor (inh). (F) Cell surface area of MEK1 inhibitor treatment datasets was measured from randomly selected 300 or more cells from three different coverslips using ImageJ software. (G) Expression of Nppa and Nppb was determined in Ad-LacZ– or Ad-Dysbindin–infected NRVCMs in the absence or presence of Cyclosporin A, a Calcineurin inhibitor, by qRT-PCR. n = 6. (H) NRVCMs overexpressing Dysbindin (Dys; or LacZ as control [cont]) were either untreated or treated with Cyclosporine A (Cyclo) and processed as in Fig. 7 B. Immunoimages were captured on Keyence microscope, and cell surface area was measured using MacroCellCount analyzer as detailed in the Materials and methods. Statistical significance was determined using two-tailed Student’s t test or by one/two-way ANOVA. Error bars show means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 50 μm.