Figure 3. LECs-secreted Reelin promotes CM proliferation and survival.

a, b, Quantitative Western blot results show increased p-AKT and p-ERK in LEC-conditioned media-treated hiPSC-CMs (a) and mouse primary CMs (b). *p=0.012 (p-AKT, a), *p=0.015 (p-ERK, a), *p=0.013 (p-AKT and p-ERK, b). N=4 (a) and N=3 (b). c, Bright field images of E17.5 Reln^ΔLEC/ΔLEC and control embryos and hearts (TAM injected at E13.5 and E14.5). Quantification of organ weight [heart (HW), liver (LW) and kidney (KW)] relative to body length (BL) indicates that hearts are smaller in Reln^ΔLEC/ΔLEC embryos. N=22 (controls) and N=11 (Reln^ΔLEC/ΔLEC) from 5 litters. *p=0.016. Controls are TAM treated Cre^- embryos and Cre⁺;Reln^+/+littermates. d-g, Double immunostaining using proliferation (EdU, pH3, Ki67 and AuroraB) and CM (cardiac Troponin C [cTnC], Prox1, αActinin and/or Mef2c) markers shows reduced CM proliferation in E17.5 Reln^ΔLEC/ΔLEC hearts. Arrows indicate proliferating CMs. h, Quantification of the immunostaining in d-g shows reduced number of EdU+, Ki67+, AuroraB+ and pH3+ CMs in E17.5 Reln^ΔLEC/ΔLEC hearts. N = 4 embryos/genotype from 3 separate litters. *p=0.02 (EdU), *p=0.01 (Ki67), **p=0.001 (pH3) and *p=0.035 (AuroraB). i, Active Caspase-3 immunostaining shows increased CM apoptosis (arrows) in E17.5 Reln^ΔLEC/ΔLEC hearts. Right panel shows quantification of the percentage of active caspase-3+ CMs in E17.5 control and Reln^ΔLEC/ΔLEC hearts. N = 4 embryos/genotype from 3 separate litters. **p=0.002. Control embryos are TAM treated Cre^- embryos and Cre⁺;Reln^+/+ littermates. Data are presented as Mean ± S.E.M. p values were calculated by unpaired two-tailed Student’s t test. n.s, not significant. Scale bars, 1 mm (c), 25 μm (d-g, i). Lower magnification for panels d-f and i are included in Supplementary Fig 2. For western blot source data, see Supplementary Fig 6 and 7.