Extended Data Figure 5. Pathways related to cell cycle are downregulated in E17.5 Prox1ΔLEC/ΔLEC embryos and LECs-conditioned media promotes CM proliferation and survival in vitro.
a, GSEA shows downregulation of cell cycle pathways and upregulation of cell death pathways in Prox1ΔLEC/ΔLEC hearts. N=4/genotype from the same litter. b, qPCR analysis confirmed the upregulation of pro-apoptotic genes (Bcl12ll, Pdcd4, Trp53ip, Stat1 and P21) and downregulation of cell cycle related genes (Cdc6, E2f1, Pcna, Mcm5 and Ccne2) in Prox1ΔLEC/ΔLEC hearts. N=3/genotype from the same litter. TAM was injected at E13.5 and E14.5. Control embryos are TAM treated Cre- embryos and Cre+;Prox1+/+ littermates. *p=0.02 (Bcl12ll), **p =0.001 (Pdcd4), 0.005 (Trp53ip), *p =0.01 (Stat1), 0.03 (P21), 0.04 (Cdc6), 0.02 (E2f1), 0.01 (Pcna), 0.02 (Mcm5) and 0.03 (Ccne2). c, Co-immunostaining against the proliferation marker Ki67 and the CM markers α-Actinin and Prox1 shows that LECs-conditioned media increases primary CM proliferation. Arrows indicate proliferating CMs. Percentage of CM proliferation was quantified by the number of Ki67+ Prox1+ CMs relative to total number of Prox1+CMs. N=3. **p=0.001. d, Co-immunostaining against the apoptotic marker active Caspase-3 and the CM markers α-Actinin and Prox1 shows reduced primary CM apoptosis upon LEC-conditioned media treatment under CoCl2 induced hypoxia. Arrows indicate apoptotic CMs. Percentage of apoptotic CMs was quantified by the number of active Caspase 3+ CMs relative to Prox1+ CMs. N=3. **p=0.003. 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 bar, 25 μm (c,d).