Role of Dlk1 in Myogenesis and Satellite Cells Figure 3. Muscle-specific Dlk1 cKO results in increased inflammation and NF-kB signaling after injury. A–B: NF-kB pathway is up- regulated in cKO injured muscle as indicated by increase DNA binding by nuclear NF-kB (A) and phosphorylated Ik-Ba (B). C: CD68 (macrophage marker) mRNA levels are increased in cKO injured muscles (n = 4). D–E: Pro-inflammatory cytokines IL-1b (D) and TNF-a (E) are up-regulated in cKO injured muscles (n = 4). doi:10.1371/journal.pone.0015055.g003 addition, the percentage of cells expressing Pax7 was not affected in the cKO compared with the wildtype cells (Fig. 4J). However, a significant reduction of cells expressing MyoD was observed in the cKO cells (Fig. 4J), leading to a shift in the cell fate status. Specifically, the proportion of self-renewing cells (Pax7+/MyoD 2 ) was increased in the Dlk1 cKO while proliferating cells (Pax7+/ + MyoD ) were decreased (Fig. 4K). These results suggest that the lack of Dlk1-initiated signaling promotes satellite cell fate choice towards the self-renewal state as the expense of proliferation. Over-expression of Dlk1 promotes myoblast cell cycle withdrawal and differentiation To directly test how Dlk1 regulates myoblasts, we over- expressed the membrane-bound form of Dlk1 in C2C12 and primary myoblasts using the Neon transfection system (Invitrogen, Inc.), which gives 50–75% transfection efficiency in our hands. We first over-expressed Dlk1 in C2C12 myoblasts (Fig. 5A–B). Strikingly, C2C12 cells over-expressing Dlk1 failed to expand and the cell number at day 3 after transfection was only 30% that of the GFP (N1-GFP, Clontech) transfected control (Fig. 5C). To confirm that Dlk1 is indeed over-expressed, we measured the mRNA and protein levels of Dlk1 in control and Dlk1 transfected cells. Dlk1-transfected C2C12 cells expressed 230 times more mRNA and much higher protein levels of Dlk1 compared to control cells transfected with GFP (Fig. 5D–E). Similar reductions in cell numbers were observed in satellite cell-derived primary myoblasts over-expressing Dlk1 (Data not shown). To investigate if the observed reduction in cell number is due to an inhibition of cell proliferation, we co-transfected primary myoblasts with 4:1 ratio of Dlk1 and GFP plasmids and measured cell proliferation with Ki67, a well-established cell proliferation marker (Fig. 5F). Notably, Ki67 expression was found to be rarely co-localized to the GFP+ cells (Fig. 5F), which should also be positively transfected with Dlk1 due to the much higher (4x) concentrations of Dlk1 plasmids used during transfection. Quantification showed that the percentages of Ki67+ cells in GFP+ cells was only 17.5%, compared to 46.2% in GFP2 cells (Fig. 5G). To further confirm this observation, we examined Ki67 expression in primary myoblasts electroporated with empty plasmid (Fig. 5H) or Dlk1 plasmid (Fig. 5I). We found that the average intensity of Ki67 immunofluorescene (normalized to DAPI fluorescence) is significantly reduced in the Dlk1 over- expressing cells (Fig. 5J). These results demonstrate that Dlk1 over- expression inhibits myoblast proliferation. Strikingly, primary myoblasts over-expressing Dlk1 displayed accelerated differentiation kinetics. At confluent cell density, it typically takes 4 days before primary myoblasts are fully differentiated. At 2 days after induced differentiation by serum withdrawal, only few myotubes were detectable in the control cells (Fig. 5K), whereas Dlk1 over-expressing cells readily differentiate and numerous myotubes were detectable at the same time point (Fig. 5L). Additionally, the average size of the myotubes formed was significantly larger in the Dlk1 over-expressing cells (Fig. 5M), PLoS ONE | www.plosone.org 5 November 2010 | Volume 5 | Issue 11 | e15055