(A-B) Increased expression of Bmp10 transcripts is detected in the DTG myocardium overexpressing Tbx20 by ISH (arrows in A, B). (C) Observed differences in gene expression was confirmed by qRT-PCR analysis of Bmp10 mRNA levels in E14.5 DTG hearts relative to levels in STG controls, which were set to 1.0 (n = 3). (D-G) The presence of pSmad1/5/8 was assessed by immunofluorescence and confocal microscopy. pSmad1/5/8 immunoreactivity (green nuclei) is increased in MF20 positive cardiomyocytes (red) and throughout the ventricles overexpressing Tbx20 in both E14.5 (arrowheads in E) and E17.5 (arrowheads in G) DTG hearts, compared to the littermate STG controls (arrowheads in D and F, respectively). (H-I) Quantitation of pSmad1/5/8 protein by Western blot (n = 6) demonstrates a 2.1-fold up-regulation in E14.5 DTG hearts, compared to littermate STG controls. Statistical significance was determined by Student’s t test, where * denotes p < 0.05.