Figure 3: Fat4 modulates Hippo signalling.
From: Amotl1 mediates sequestration of the Hippo effector Yap1 downstream of Fat4 to restrict heart growth
(a) Relative expression of Yap1 target genes in Fat4+/+ (n=5), Fat4+/− (n=5) or Fat4−/− (n=7) hearts at P0. *P<0.05 (analysis of variance (ANOVA)). (b) Immunodetection of Yap1 localization in the heart of the indicated genotype at P0. Nuclei are encircled with dashed white lines. (c) Immunodetection of Yap1 localization in primary cultures of neonatal rat cardiomyocytes treated with the indicated siRNA. (d) Quantification of the increased percentage of nuclei with a strong Yap1 signal when Fat4 is downregulated (n=6 cultures) relative to the control (n=6). **P<0.01 (Student test). (e) Immunodetection of Yap1 (nuclear, arrows) localization in primary cultures of cardiomyocytes transfected with the indicated plasmids. Fat4-ΔECD-Flag, which is depleted for the extracellular domain (ECD), is used for overexpression. (f) Quantification of its nuclear to cytoplasmic localization, which is decreased when Fat4 is overexpressed (n=14 cells) relative to control GFP (n=9). ***P<0.001 (Student test). (g) The percentage of proliferating Ki67-positive cardiomyocytes, counted by flow cytometry from primary cell cultures, indicates a genetic rescue of Fat4 siRNA by Yap1 siRNA (n=20 ctrl, 20 Fat4, 8 Yap1 and 8 Fat4+Yap1 siRNA cultures). ***P<0.001 (ANOVA). ns, no significant difference. Scale bars: 5 μm in b, 20 μm in c, 10 μm elsewhere.
