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Fig. 3 Hedgehog, Igf and Tgfβ signaling are required for myocardial regeneration in zebrafish. (A) The Shh ligand shha (transgenic reporter) and regulator/response gene ptch2 (transgenic reporter), Igf ligand igf2b (in situ hybridization) and receptor Igfr1 (immunofluorescence), and Tgfβ ligand tgfb3 (in situ hybridization) each showed increased expression in the wound area by 7 dpa. Brackets indicate injury site. Scale bar: 100 μm. (B) Treatment with CyA (10 μM) or NVP AEW541 (2 μM) from 6 to 7 dpa decreased cardiomyocyte proliferation. Mef2, red; PCNA, green. Brackets indicate injury site. Insets: High magnifications of the boxed areas. Arrowheads indicate proliferating cardiomyocytes. Scale bar: 50 μm. (C) Quantification of the effects of CyA (10 μM), NVP AEW541 (2 μM) and SB-431542 (10 μM) on cardiomyocyte proliferation after resection injury. Fish were treated from 6 to 7 dpa. n=9-15, mean±s.e.m. *P<0.01, Student′s t-test. (D) Quantification of the effects of CyA, NVP AEW541 and SB-431542 on proliferation after genetic cardiomyocyte ablation. Fish were treated from 6 to 7 dpa. n=12-13, mean±s.e.m. *P<0.001, Student′s t-test. Data in each graph were obtained from different clutches of fish.