Figure 4.

p90RSK activation in mouse aortic arch and in MECs under hyperglycemia. (A) Confocal images of the endothelium of the greater curvature in mouse aortic arch of twelve-week-old C57BL/6 wild type. The en face specimen was double-stained with anti-VE-Cadherin (green) and anti-phospho-p90RSK (upper two panels, grey and red (merge)) or anti-total p90RSK (lower two panels, grey and red (merge)). Individual red signals as well as merged images are shown as indicated. Two weeks after STZ injection, blood glucose level was measured. Insulin (Ins) (Humalin N, twice daily, 5IU/kg) treatment was started after 3 days of STZ injection. Scale bars, 50µm. (B) Glucose tests in the blood of male mice after insulin treatment followed by injection of vehicle or STZ. (C, D) En face confocal images of diabetic and non-diabetic mice in s-flow area (greater curvature of aortic arch area) were obtained using the same image acquisition setting, and fluorescence intensity was quantified. Data are shown as means ± SEM. (E) C57BL/6 wild type mice were intra-peritoneally injected for three days with vehicle or STZ (100mg/kg/day), followed by vehicle or FMK-MEA injection as described in Fig.5A. After four days of FMK-MEA injection, MECs were isolated from the lungs of these mice as described in methods. Immediately, RIPA buffer was added to lyse the cells, then p-p90RSK (upper) and total p90RSK (lower) protein expressions were assayed by Western blotting using each specific antibody (n=3).