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Fig. 8 Decreased Tgfβ1 expression is observed in injured ccn2a mutant heart and Tgfβ/pSmad3 inhibition recapitulates the genetic regulation by ccn2a in injured heart. (A) Maximum intensity projection of confocal images of 10 μm sagittal cryosection of wild-type and ccn2a−/− heart at 4 dpci, expressing DsRed in CM nuclei (magenta), immunostained for Tgfβ1 (cyan) and stained for F-actin (yellow; marks all cells). Arrowheads indicate Tgfβ1 expression. Dotted lines mark the injury edge. (B) Percentage of relative fluorescence intensity of Tgfβ1 (n=4 each). The heart section from each heart showing the highest fluorescence intensity for Tgfβ1 was considered for quantification, and the mean of the wild-type control value was set to 100%. (C) Schematic of the experimental procedures used in D,E. (D,E) qPCR-based quantification of gene expression from DMSO- or SB431542 (TGFβ type I receptor inhibitor)-treated animals at 4 dpci (n=3, each sample is a pool of six hearts). The mean value of the DMSO-treated control for each gene was set to 1. (F) Schematic of the experimental procedures used in G,H. (G) Maximum intensity projections of confocal images of 7 dpci whole-mount heart expressing DsRed in CM nuclei and EGFP in endothelial/endocardial cells. Arrowheads indicate infiltrated CMs in the injured tissue. Dotted lines indicate the injury edge. (H) Analysis of infiltrated CMs in injured cardiac tissue quantified four DMSO-treated and four SB431542-treated wild-type animals from two independent experiments. The mean of the DMSO-treated control value was set to 100%. Data are mean±s.d. (I) Model of the regulatory role of Ccn2a in heart regeneration. CM, cardiomyocyte; ECM, extracellular matrix. The statistical significance of differences was evaluated by a two-tailed Student's t-test (GraphPad Prism). Mean Ct values are provided in Table S4.