PMC

Inhibition of PKCβ (DIF-Let-7e+PKCβi) provokes a recovery in the expression of these genes. Levels were normalized to GAPDH and data represent the mean from three independent experiments ±S.D (*) P<0.05 versus DIF, (+) P<0.05 versus DIF-Let7e. Groups: differentiated EBs+control antimiR (DIF), Non treated EBs (CONTROL), differentiated EBs+LNA-antiLet-7e (DIF-Let-7e), differentiated EBs+LNA-antiLet-7e and PKCβ inhibitor (Gö 6983) (DIF-Let-7e+PKCβi), differentiated EBs+BIO (DIF+BIO).

Downregulated expressions of both GSK3β^P and β-catenin were detected in differentiated EBs and the expressions were recovered when LNA-antiLet-7e (DIF-Let-7e) was added. Inhibition of PKCβ in the differentiated EBs (DIF-Let-7e+PKCβi) provokes a downregulation again. Western blots of β-actin as loading controls are shown. n = 3 (*) P<0.05 versus DIF, (+) P<0.05 versus versus DIF-Let7e. Groups: differentiated EBs+control antimiR (DIF), Non treated EBs (CONTROL), differentiated EBs+LNA-antiLet-7e (DIF-Let-7e), differentiated EBs+LNA-antiLet-7e and PKCβ inhibitor (Gö 6983) (DIF-Let-7e+PKCβi).

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). A) 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) 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. C) 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.

A) Total RNA was extracted from EBs cultured for 1 to 10 days with ATRA and activin A [black columns] or without factors [white columns] and assayed for gene specific expression (Pax2, WT-1, Wnt4, Wnt9b and Notch2) by RT-PCR. Data shown starts on day 5, the time when expression of genes tested starts to be significantly upregulated. n = 4. Data are means ±S.D [*] P<0.05 vs. non treated EBs (white columns) and normalized to GAPDH; B) Western blot analysis of Pax2 and WT1 in cell lysates of EBs cultured for 8 days. Upregulated expressions of both proteins were detected in differentiated EBs. Western blots of β-actin as loading control are shown. C) miRNAs microarray in EBs cultured for 8 days with ATRA and activin A (DIF EBs) shows a positive fold difference >1.5 for all the family members of let-7 miRNAs tested (black columns), and in particular a (>9) fold difference for the miRNA let-7e (*) versus EBs cultured for 8 days without factors (CONTROL EBs). 88 different miRNAs were analyzed, but only the upreglated ones are shown. miRNA expression was normalized to the average of the four small nuclear housekeeping RNA (snoRNA251, snoRNA202, snoRNA142 and U6 snRNA). The fold difference for each miRNA was calculated using ΔΔCt method [ΔΔCt = ΔCt [DIF EBs] – ΔCt [CONTROL EBs]. D) Northern Blot probed for the mature miRNA let-7e in EBs cultured for 8 days with ATRA and activin A (DIF) or without factors (CONTROL) confirmed the result of the microarray. Result is graphed in Arbitrary units of optical density (O.D) n = 3±S.D [*] P<0.05 vs. CONTROL.