Fig. 3.

Activation of STAT3 following I/R injury. NRVMs were subjected to 4 h ischaemia or ischaemia plus the indicated times of reperfusion and lysates were subjected to Western blotting using antibodies for (A) tyrosine and serine phosphorylated STAT3 or (B) tyrosine and serine phosphorylated STAT1 and caspase-9. GAPDH was used as a loading control. (C) STAT3 dependent luciferase reporter activity is enhanced by I/R injury. 1 μg of Ly6E luciferase reporter and 0.1 μg CMV-renilla were transfected into NRVMs in 6-well plates for 24 h. Cells were then subjected to 4 h ischaemia and 24 h reperfusion (I/R) and luciferase activity was measured ^∗∗∗p < 0.001, n = 3, experiment repeated in duplicate. (D) STAT3 dependent gene expression is increased by I/R injury. NRVMs were subjected to a time course of I/R injury and the levels of SOCS3 and c-Fos were measured by qPCR and normalised to control (con) levels. Isc = Ischaemia alone without reperfusion. Statistical analysis was carried out using a one-way ANOVA with Dunnett’s post test, n = 3 samples, experiments repeated in triplicate, ^∗p < 0.05, ^∗∗p < 0.01 compared to con. (E) NRMVs were transduced with STAT3C adenovirus at MOI = 100. After 24 h, the expression of SOCS3 and c-fos and was examined by qPCR. ^∗∗p < 0.01, Student’s t-test, n = 3 per group, repeated in duplicate.