Fig. 5.
Inhibition of miR-27b activity in adult Pax3GFP/+ satellite cells delays differentiation. (A) Detection of Myogenin (MyoG) (green) and Troponin T (TropT) (red) by immunocytochemistry with the corresponding antibodies after satellite cells were transfected with an anti miR-27b inhibitor or control oligonucleotide and cultured for 4 days. DAPI staining of nuclei is shown in blue. Some MyoG-positive cells can be detected together with a low number of mononucleated TropT-positive cells. (B) The number of positive cells for these markers is reduced after transfection with anti miR-27b inhibitor, whereas the total number of DAPI-positive nuclei increases. (C) An equivalent series of results in satellite cells from Pax3GFP/flox mice that lack Pax3. (D) Immunocytochemistry as in A at 5 days shows that MyoG expression is normally reduced as cells differentiate into polynucleated, TropT-expressing myotubes. In contrast, cells transfected with anti-miR-27b inhibitor have higher MyoG expression and lower TropT expression (D), indicating that differentiation is delayed in response to inhibiting the activity of miR-27b. (E) Quantitation of these results as in B. At 5 days, only MyoG-positive nuclei were counted because TropT expression was found in the cytoplasm of polynucleated fibers at this stage. Results in B, C, and E are indicated relative to transfection with the negative control oligonucleotide and standard deviations are reported from 3 independent transfections, with P < 0.05 using paired Student's test. (F) Fusion index, estimated as the number of DAPI-positive nuclei per myofiber in control or anti-miR-27b inhibitor-treated satellite cell cultures. Results are averages with standard deviations from 3 independent transfections, with P < 0.05 using paired Student's test.