Ectopic miR-181a expression in primary LECs results in reduced Prox1 mRNA and protein levels. (A) Prox1 mRNA expression is substantially reduced in primary embryonic LECs after transfection of miR-181a, compared with transfection with a negative control (miR-neg) that has no established targets; n = 6. *P < .001. (B) Immunostaining of primary embryonic LECs transfected with control miR-neg (top panel) versus miR-181a (bottom panel), illustrating dramatic reduction in nuclear Prox1 as a result of miR-181 activity. CD31 (green) and Prox1 (red). Scale bar represents 100 μm. (Ci) Western blot illustrating that Prox1 protein is substantially reduced relative to β-actin in primary embryonic LECs after the introduction of miR-181a. (Cii) Quantification of reduction in Prox1 protein levels relative to β-actin levels in primary embryonic LECs after ectopic miR-181a expression, compared with miR-neg expression. Data are representative of 3 independent experiments. (Di) Dose-response analysis of Prox1 mRNA expression in primary embryonic LECs after transfection of increasing concentrations of miR-181a (black bars), compared with transfection with negative control miR-neg (gray bars). Significant reduction in Prox1 mRNA and protein levels is achieved at 5nM, the lowest level tested; n = 4. *P < .05. Data are mean ± SEM. P values were calculated using Student paired t test. (Dii) Prox1 mRNA and protein levels as assessed by Western blot are shown relative to the negative control.