Figure 5.

Effect of miR-146a on the expression of Notch signaling molecules in human primary LECs identified by western blot and immunostaining. (A,B) Total extracted protein from transfected LECs with miR-146a mimic (M) or its inhibitor (I) and their corresponding controls, mimic control (MC) and inhibitor control (IC), respectively, was separated on gradient SDS-PAGE gels, transferred to membrane, and probed with Notch signaling molecule antibodies (Table 2). Antibody to β-actin was used as loading control and for semi-quantitation. (A) miR-146a treatment increased, whereas its inhibitor decreased, the protein level of Notch-1. On the contrary, miR-146a treatment decreased, whereas its inhibitor increased Numb expression level in primary LECs compared to their corresponding controls. (B) Overexpression of miR-146a in transfected cells decreased, whereas its inhibition increased the protein level of Notch-2 compared to their corresponding controls. However, there was no significant change in Hes1 expression level in treated cells compared to their corresponding controls. The bar graph represents average ± SEM (Standard Error of the Mean) of pooled values (n = 4) of densitometric scans. * p < 0.05, compared with scrambled control values by paired two-tailed t test. Each bar represents the changes of mimic or inhibitor compared to their corresponding controls, shown as M/MC and inhibitor (I)/inhibitor control (IC). (C) Effect of miR-146a on Notch signaling molecule expressions in human primary LECs by immunostaining. Primary human LECs transfected with miR-146a mimic showed increased expression of Notch-1 and decreased expressions of Numb and Notch-2, whereas there was no significant change in Hes1 expression level in miR-146aM-treated cells compared to the mimic control. The same exposure time was used for each set of compared immunostained sections; the pictures are representative of three independent experiments of each transfected primary LEC (n = 3).