(A) qPCR analysis to show fold changes in Scx expression in avian valve precursor cells, and C3H10T1/2 and NIH3T3 murine fibroblast cell lines treated with 200pM Tgfβ2 for 48 hours compared to BSA vehicle treated controls (n=3). (B) qPCR to show Scx expression in E13.5 hearts from Tgfβ2+/− and Tgfβ2−/− mice compared to wild type (Tgfβ2+/+) littermate controls. (C–D) Immunohistochemistry to detect CSPG expression (red) in avian valve precursor cell cultures with BSA vehicle or 200pM Tgfβ2 treatment for 48 hours. (E–F) Immunohistochemistry to detect CSPG expression (red) in porcine VIC cultures treated for 48 hours with BSA vehicle or 200pM Tgfβ2. Blue indicates DAPI-positive cell nuclei. (G, H) Quantitation of CSPG immunoreactivity in avian valve precursor cells (C, D) and porcine VICs (E, F) treated with 200pM Tgfβ2 compared to BSA control. (*=p<0.05 using oneway ANOVA plus a post-hoc test n=3.) (I) qPCR to show fold changes in Aggrecan expression in mitral valve explants from Scx+/+ and Scx−/− PND1 pups treated with Tgfβ2 treatment or PBS vehicle for 48 hours. (*=p<0.05 Tgfβ2 versus PBS, #=p<0.05 Tgfβ2 treatment in Scx+/+ versus Scx−/− using Students t-test, n=3).