Figure 1: Myc induces global RNA production in cardiomyocytes.

(A) Endogenous Myc protein in cardiomyocytes transfected with siRNA directed against Myc or with control siRNA (C) was detected by western blotting (upper panels). Adenovirally transduced Myc (Ad-Myc) in cardiomyocytes with increasing MOI (multiplicity of infection) was detected by western blotting (lower panels). Gapdh protein was used as a loading control. Before harvesting, cells were treated with 5 μM MG132 for 1 h. (B) Levels of overexpressed Myc protein in cardiomyocytes with increasing MOI normalized to Gapdh protein levels were calculated using imaging software (ImageQuantTL (GE), n = 3, mean ± SD). NTD: non transduction control. *p < 0.05, ^#p < 0.01 vs NTD. (C) Algorithm for quantifying cardiomyocyte-specific nuclear EU-labeled RNA. First, cardiomyocytes were distinguished as troponin I–positive cells before their cell areas were segmented by locating the nuclei. EU images merged with nuclei specifically in cardiomyocytes were extracted and calculated as synthesized nuclear RNA per cardiomyocyte. Methodology details are shown in Fig. S1D. (D) Cardiomyocytes seeded in 96 well plates were transduced with either Ad-LacZ or Myc at MOI (multiplicity of infection) 20. Twenty-four hours after transduction, cells were labeled with EU for either 1 or 3 h before being fixed and immunostained. Representative images are shown. Scale bar: 50 μM. (E) Cardiomyocytes were transduced with either Ad-LacZ, Myc, or MycΔC at increasing MOI. Twenty-four hours after transduction, cells were labeled with EU for 1 h before being fixed and immunostained. Average nuclear EU intensities normalized to the cardiomyocyte cell number were calculated (n = 3, mean ± SD). *p < 0.05, ^#p < 0.001 vs NTD.