Figure 1: YAP interacts with FoxO1 and regulates its transcriptional activity.

(a) The localization of FoxO1 and YAP was examined. Myocytes were stained with FoxO1 (red), YAP (green) and DAPI (blue). Scale bar, 10 μm. (b) Cells were stained with Rabbit anti-FoxO1 antibody and/or Mouse anti-YAP antibody, and in vivo protein–protein interaction between FoxO1 and YAP (red dots) was detected with secondary proximity probes, anti-Rabbit-PLUS and anti-Mouse-MINUS, using the Duolink in situ PLA detection kit. Scale bar, 10 μm. (c) Neonatal cardiomyocyte lysates were prepared and immunoprecipitated with FoxO1 antibody or control IgG. The interaction between FoxO1 and YAP was examined. Immunoblots of input lysate controls (5% of inputs) are also shown. (d) Recombinant GST-YAP and myc-FoxO1 were used to examine the direct interaction between YAP and FoxO1. (e) Cardiomyocytes were co-transfected with 1 μg FoxO1-luc vector and the indicated expression vectors. After 48 h, the luciferase activity was measured, demonstrating that YAP enhanced FoxO1 transcriptional activity (*P<0.05 versus empty vector, n=3). (f) Overexpression of Lats2 inhibited, whereas knockdown of Lats2 enhanced, FoxO1 activity. Cardiomyocytes were transfected with 1 μg FoxO1-luc vector. After 6 h, myocytes were transduced with the indicated virus and luciferase activity was measured (*P<0.05 versus LacZ or sh-con, n=3). Data are shown as mean±s.e.m. P values were determined using unpaired Student’s t-test.