Fig. S6

Fig. S6 Chemical Screening for Regulators of Epicardial Cell Proliferation (Related to Figure 7)
(A) Whole mounted tcf21:nucEGFP ventricles after 6 days of culture as explants, treated with DMSO (Vehicle) or GSK1007102B. Framed regions are enlarged and shown below.
(B) Quantification of EGFP fluorescence density. n = 8 (Vehicle) and 5 (GSK1007102B) explants, respectively. *** P < 0.001, Student’s t-test. Bars indicate mean ± S.D.
(C) Images of fixed and flattened tcf21:nucEGFP explants after 2 days of culture with DMSO (Vehicle) or SB431542. Samples were incubated with EdU for 1 h before fixation (shown in red and grayscale). DNA was stained with DAPI (blue). Scale bars, 100 μm.
(D) Quantification of EGFP+EdU+ nuclei per mm2 on the ventricular surface, from hearts in (C). n = 3 explants for each. * P < 0.05, Student’s t-test. All error bars indicate S.D.
(E, F) PI3K regulates epicardial regeneration. (E) Schematic for experiments in (F). tcf21:NTR; tcf21:nucEGFP animals were used. LY294002 (50 μM) was added for 8 days (3 - 11 dpi). Then, explants were rinsed twice with PBS and further cultured for 4 days. (F) Epicardial regeneration ex vivo in the presence of DMSO (Vehicle) or 50 μm LY294002. All vehicle-treated explants (16 of 16) displayed regeneration. All LY294002- treated explants displayed a block of regeneration (23 of 23), which resumed in each case after drug wash-out. Scale bar, 200 μm.