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

<p><b>A–B</b>: <i>Dlk1</i> over-expression in C2C12 myoblasts. Cells were transfected with either GFP (<b>Control</b>; <b>A</b>) or <b>Dlk1 OE</b> (<b>B</b>) plasmids and cultured for 3 days. C: Cell numbers of <b>Control</b> and <b>Dlk1 OE</b> at day 3 after transfection. <b>D–E</b>: Relative <i>Dlk1</i> mRNA levels in the <b>Control</b> and <b>Dlk1 OE</b> cells measured by quantitative Realtime PCR (n = 4). <b>E</b>: Protein levels of Dlk1, GFP and α-tubulin in GFP control transfected cells (<b>GFP</b>), Dlk1 over-expression cells (<b>DLK1</b>), and cells transfected with an unrelated negative control gene (<b>TSG101</b>). <b>F</b>: 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. <b>G</b>: Percentage of cells displaying Ki67 staining in <b>Control</b> and <b>Dlk1 OE</b> cells (n = 3). <b>H–J</b>: Primary myoblasts transfected with an empty plasmid (H, control) or Dlk1 plasmid (<b>I</b>, <b>OE</b>) 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 (<b>J</b>, n = 3 per treatment). <b>K–M</b>: Phase-contrast images of primary myoblasts differentiated for 48 hrs after transfected with either empty plasmids (<b>K</b>) or Dlk1 OE plasmids (<b>L</b>). <b>M</b>: Average pixel size of myotubes in <b>Control</b> and <b>Dlk1 OE</b> treatments as shown in <b>H&I</b> (n = 20 random tubes measured). <b>N–O</b>: Primary myoblasts grown on Matrigel plus vehicle control (<b>N</b>) and on Matrigel plus Dlk1 recombinant protein (<b>O</b>) after 3 days in differentiation medium. <b>Green</b> fluorescence (<b>MF20</b>) marks sarcomere myosin heavy chain and <b>Blue</b> is <b>DAPI</b> counterstaining for nuclei. <b>P</b>: The relative diameters of the resulting myotubes as shown in <b>N & O</b> 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.</p>