miRNA let-7e silencing reduces the expression of the differentiation markers WT1, Pax2, Wnt4 and increases the protein levels of PKCβ.

<p>Differentiated EBs were collected on day 10 and LNA-antiLet-7e was added on days 7 and 9 of differentiation to collect the cells after 24 h of the last transfection (day 10). Groups: Non treated EBs (CONTROL), differentiated EBs+control antimiR (DIF), differentiated EBs and miRNA let-7e silenced (DIF-Let-7e). <b>A)</b> RT-PCR of the mature miRNA let-7e confirms that miRNA let-7e is silenced after the administration of antiLet-7e. n = 3. Data are means ±S.D (*) P<0.05 vs. DIF and normalized to U6 snRNA; <b>B)</b> RT-PCR of WT1, Pax2 and Wnt4 show that miRNA let-7e silencing provokes a downregulation of these genes. Levels were normalized to GAPDH and data represent the mean of three independent experiments ±S.D (*) P<0.05 versus CONTROL, ±S.D (+) P<0.05 versus DIF. <b>C)</b> PKCβ levels evaluated by Western blot analysis show a downregulated expression in differentiated EBs (DIF) and a recovery of the expression in silenced EBs (DIF-Let-7e). Upper panel: Western blot analysis of PKCβ. Lower panel: Western blot analysis of β-actin.