Figure 5
Smad3 and Smurf1, but not Fols2, are essential for miR-195 driven cardiomyogenesis in PE/ST explants. Panel A. qPCR analyses of Smad3, Smurf1 and Fols2 in control, siRNA treated and siRNA plus miR-195 overexpression. Observe that siRNAs against Smad3, Smurf1 and Fols2 significantly decrease Smad3, Smurf1 and Fols2, respectively. Observe also that miR-195 administration can rescue Smad3 and Smurf1 up-regulation but not Fols2. Panel B. qPCR analyses of cardiomyogenic markers in control, siSmad3 treated and siSmad3 plus miR-195 overexpression. Observe that expression of Mef2c, Nkx2.5, Gata4 and Tnnt2 is severely impaired in both siSmad3 treated and siSmad3 plus miR-195 overexpression conditions. Panel C. qPCR analyses of cardiomyogenic markers in control, siSmurf1 treated and siSmurf1 plus miR-195 overexpression. Observe that expression of Nkx2.5, Gata4 and Tnnt2 is severely impaired in both siSmurf1 treated and siSmurf1 plus miR-195 overexpression conditions. Panel D. qPCR analyses of cardiomyogenic markers in control, siFols2 treated and siFols2 plus miR-195 overexpression. Observe that expression of Mef2c, Gata4 is not altered, Nkx2.5 is diminished but Tnnt2 is significantly increased in siSmurf1 conditions. Furthermore, Mef2c, Nkx2.5, Gata4 and Tnnt2 expression is significantly increased in siSmurf1 plus miR-195 overexpression conditions. HH17 PE were dissected from >30 embryos, treated with the corresponding microRNA mimics, siRNA or the combination of both (microRNA mimic and siRNA) and subsequently pooled to performed RNA isolation. On each case, three distinct biological replicates were subsequently tested by qPCR.
