PKG and PKC Are Down-Regulated during Cardiomyocyte Differentiation from Embryonic Stem Cells: Manipulation of These Pathways Enhances Cardiomyocyte Production
Gene expression was measured by semiquantitative RT-PCR using PKG1-specific primers. (a) Beating and nonbeating areas of similar sizes were dissected apart as described in [4, 10], followed by RNA isolation and quantification. Control GAPDH RT-PCR amplification revealed that 35 cycles were in the linear range and nucleotides in the PCR reaction were not exhausted. (b) After normalization to GAPDH levels, PKG1 mRNA levels were lower by approximately one-half in beating areas when compared to nonbeating areas (pixel intensity = 70 11.0 to 139 19.1, resp.). A representative ratio of PKG mRNA signal revealed 1.98X more in nonbeating areas when compared to beating areas (inset in (b)). (c) Inhibiting PKG1 resulted in dramatic and significant () increase in the size of beating areas within EBs; however, the total size of EBs was not changed by PKG inhibition (d).