Figure 4: Expression of miR-103 in ECs during atherosclerosis.

(a,b) The expression levels of miR-103, miR-301b, miR-433 and miR-652 (a; n=5 per group) and their enrichment in AGO2-IPs of human ECs (b). The results of b are expressed as the fold enrichment of miRNAs in AGO2-IP samples compared with control IgG-IP samples. Results of one representative experiment are shown in b. (c) Quantitative RT–PCR analyses of the expression levels of miR-103, miR-301b, miR-433 and miR-652 in HAECs with and without TNF-α stimulation (n=3–4 per group). (d) MiRNA expression level in HAECs treated with vehicle or the NF-κB-inhibitor BAY11-7085 (n=4–5 per group). (e) Expression levels of miR-103 in HAECs treated with PBS, native low-density lipoprotein (nLDL) or mildly oxidized-low-density lipoprotein (moxLDL; n=4 per group). (f) Combined in situ PCR detection of miR-103 and immunostaining of the endothelial marker CD31 in carotid sections from EC-Dicer^WT and EC-Dicer^flox mice fed a HFD for 4 weeks. Representative images of three independent experiments are shown. (g) Endothelial miR-103 expression in human atherosclerotic plaques determined by in situ PCR and immunostaining of von Willebrand factor (vWF). (h) Movat’s pentachrome staining of a human atherosclerotic plaque section located adjacent to that used for the in situ detection of miR-103. The region of the plaque used for miR-103 expression analysis is indicated. Scale bars, 12 μm (f), 25 μm (g) and 500 μm (h). Asterisks indicate the lumen. The data are represented as the mean±s.e.m. of the indicated number (n) of repeats. *P<0.05, **P<0.01 and ***P<0.001 by Student’s t-test and one-way analysis of variance.