Dlk1 over-expression (OE) or recombinant Dlk1 protein inhibits proliferation but promotes differentiation of myoblasts.

A–B: Dlk1 over-expression in C2C12 myoblasts. Cells were transfected with either GFP (Control; A) or Dlk1 OE (B) plasmids and cultured for 3 days. C: Cell numbers of Control and Dlk1 OE at day 3 after transfection. D–E: Relative Dlk1 mRNA levels in the Control and Dlk1 OE cells measured by quantitative Realtime PCR (n = 4). E: Protein levels of Dlk1, GFP and α-tubulin in GFP control transfected cells (GFP), Dlk1 over-expression cells (DLK1), and cells transfected with an unrelated negative control gene (TSG101). F: Primary myoblasts co-transfected with Dlk1 and GFP (4∶1) plasmids were cultured for 2 days and labeled with a cell proliferation marker Ki67 together with GFP staining. GFP signals (indicating Dlk1 positively transfected cells) exhibit little colocalized with Ki67 immunofluorescence. G: Percentage of cells displaying Ki67 staining in Control and Dlk1 OE cells (n = 3). H–J: Primary myoblasts transfected with an empty plasmid (H, control) or Dlk1 plasmid (I, OE) cultured for 3 days in growth medium and labeled with Ki67 (in red) and DAPI (in blue). The average Ki67 immunofluorescenc intensity was measured with Photoshop and normalized to DAPI intensity (J, n = 3 per treatment). K–M: Phase-contrast images of primary myoblasts differentiated for 48 hrs after transfected with either empty plasmids (K) or Dlk1 OE plasmids (L). M: Average pixel size of myotubes in Control and Dlk1 OE treatments as shown in H&I (n = 20 random tubes measured). N–O: Primary myoblasts grown on Matrigel plus vehicle control (N) and on Matrigel plus Dlk1 recombinant protein (O) after 3 days in differentiation medium. Green fluorescence (MF20) marks sarcomere myosin heavy chain and Blue is DAPI counterstaining for nuclei. P: The relative diameters of the resulting myotubes as shown in N & O were measured with Image J software. Control is the open bar (n = 66 myotubes) and Dlk1 recombinant protein treated cells are represented by the solid black bar (n = 66 myotubes). Asterisks in all bar graphs indicate p<0.05 compared to control groups by student t-test.