The effects of HU and Akti XII on the expression of E-selectin and ICAM-1, plasma NOx levels, and AKT2 phosphorylation in TNF-α-challenged Berkeley mice. (A-D) Following intravital microscopy, the cremaster muscle was excised, fixed, and embedded in paraffin for immunohistochemistry. Sections of the muscle were labeled with rat anti-mouse E-selectin or ICAM-1 and then Dylight 488-labeled anti-rat immunoglobulin G antibodies, followed by incubation with phycoerythrin-labeled anti-PECAM-1 antibodies and a mounting reagent containing 4,6 diamidino-2-phenylindole. (A,C) Representative images of E-selectin or ICAM-1 and PECAM-1 staining. (B,D) The geometric mean fluorescence intensities (MFI) of E-selectin or ICAM-1 expression in venules of Berkeley mice. Data represent the mean ± standard deviation (SD) (n = 45-57 venules in 6-8 mice per group). (E) Following intravital microscopy, plasma NOx levels were measured as described in supplemental Methods. Data represent the mean ± SD (n = 6-8 mice per group). (F-I) Berkeley mice were treated with saline, HU, Akti XII, or both HU and Akti XII as described in supplemental Methods. Neutrophils and platelets were isolated and stimulated with fMLF and thrombin, respectively. Immunoblotting was performed by using equal amounts of protein (50 μg) followed by densitometry using Image J software. Representative blots (F,H) and quantitation of AKT2 phosphorylation after normalization to total AKT expression in neutrophils (G) and platelets (I). Data represent the mean ± standard error of the mean (n = 6 mice per group). *P < .05, **P < .01, and ***P < .001 vs vehicle control; ANOVA and Dunnett’s test. #P < .05 and ##P < .01 between two groups; Student t test.
