Fig. 3

mir-1/206-resistant Srsf9-GFP expression blunts myogenic differentiation. a C2C12 cell lines stably expressing GFP alone (GFP control) or GFP-tagged Srsf9 linked to a heterologous miR-1/206-resistant 3′ UTR (Srsf9-GFP) were differentiated for 6 days, immunostained, and imaged (20X objective). Myosin heavy chain, a marker of terminal differentiation, was visualized with the F59 antibody and an AlexaFluor568-linked secondary antibody. DNA was visualized by DAPI staining and GFP or Srsf9-GFP was visualized by GFP autofluorescence. Scale bars denote 6.5 μm. b Srsf9-GFP persistence reduces fusion. The fusion index of Srsf9-GFP cells was significantly lower than either pC-Empty cells (an empty expression plasmid) or GFP control cells. Eight to 10 non-overlapping fields of view were analyzed for each cell line. *p < 0.05 vs. pC-Empty; ^###p < 0.001 vs. GFP control. c Srsf9-GFP persistence reduces myotube size. Myotube area was assessed by calculating the myosin-positive area of D6 myotubes. The myosin-positive area/DAPI-positive area was calculated for 8–10 non-overlapping fields of view. Myosin-positive area was significantly lower than both pC-Empty and GFP control cell lines. *p ≤ 0.05 vs. pC-Empty; ^#p < 0.05 vs. GFP control. d Srsf9-GFP persistence reduces myosin mRNA expression. mRNA levels of perinatal myosin (Myh8) were assessed by qPCR at days 0, 1, 2, 4, and 6 of differentiation. Statistically different quadratic curves were fit to GFP control and Srsf9-GFP series. e Srsf9-GFP persistence reduces MyoG induction. MyoG mRNA levels were measured by qPCR as in panel (d). Statistically different quadratic curves were fit to GFP control and Srsf9-GFP. For d and e, asterisks next to the Srsf9-GFP legend denote p < 0.0001 for the curve fit comparison