Role of Dlk1 in Myogenesis and Satellite Cells Figure 5. 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 a-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 PLoS ONE | www.plosone.org 7 November 2010 | Volume 5 | Issue 11 | e15055