Fig. 5

miR-27b controls of Dll4 in ischemic tissue and in vascular endothelium. Five-μm crosswise tissue sections of calf muscle or tumor tissue were stained for Dll4, a miR-27b target (green). (a) Representative photomicrographs of Dll4 immunostaining of the calf muscle sections. (b) Total Dll4 positive area was quantified and adjustment made for fluorescence intensity using Elements software (Nikon). Note increased Dll4 immunostaining under ischemic conditions in animals treated with control RNAi (NC), and significant attenuation by miR-27b (27b). The data are presented as box plot with whiskers. Median values are shown. Statistical significance and P values were determined using Wilcoxon Signed Rank test. (c, d) Sections of tumors from untreated animals, animals treated with control RNAi (NC), miR-27b mimic (M) or inhibitor (I) were stained for Dll4 (green) and total immunofluorescence measured in at least 8 sections/condition using Nikon Elements software. Data is presented as box plot with whiskers and mean values are shown. Statistical significance (P values) was determined by Wilcoxon test as above. (e) Dual immunofluorescence for Dll4 (green) and CD31 (red), to determine vascular-specific expression of Dll4. Dual positivity appears as yellow. (f) Vascular-specific Dll4 expression (a number of Dll4-positive capillary structures/field) was measured using Nikon Elements software and statistical significance calculated as above. P values are shown