Figure 4: Hippo activation stimulates oxidative stress in response to I/R.

(a) Representative immunoblots indicating that I/R activated Hippo signalling. Left, WT mice were subjected to ischaemia for 45 min followed by 2 h of reperfusion. Sham and ischaemic area samples were used for immunoblot analysis for p-Lats2 (T1041 and S872), Lats2, p-Mst1 (Thr183) and Mst1. Right, Tg-DN-Lats2 and control WT mice were subjected to ischaemia for 45 min followed by 2 h of reperfusion. Sham and ischaemic area samples were used for immunoblot analysis for p-YAP (S127), YAP, Lats2 and GAPDH. The enhancement in YAP phosphorylation during I/R was attenuated in Tg-DN-Lats2, confirming that Lats2 activation was suppressed. (b) Tg-DN-Lats2 and control WT mice were subjected to ischaemia for 45 min followed by 2 h of reperfusion. The cytosolic and nuclear fractions of heart homogenates were prepared and subjected to immunoblot analysis of p-YAP (S127), YAP, Lats2, GAPDH and Lamin A/C (SE, short exposure; LE, long exposure). (c,d) Activation of Lats2 decreased antioxidant protein levels and enhanced oxidative stress during I/R. Tg-DN-Lats2 and control WT mice were subjected to ischaemia for 45 min followed by 24 h of reperfusion. (c) Sham and ischaemic area samples were used for immunoblot analysis for catalase, MnSOD, and α-tubulin. (d) The antioxidant capacity of the myocardium was examined (*P<0.05 versus WT/sham, #P<0.05 versus WT/I/R, n=4). Data are shown as mean±s.e.m. P values were determined using one-way ANOVA followed by a Newman–Keuls comparison test.