Dlk1 and myostatin display an inverse relationship in human myoblasts, and the expression of Dlk1 is observed in human rhabdomyosarcoma cell lines.

<p>A: Expression of Dlk1 and myostatin in two human myoblast cultures during G₀ arrest, proliferation before and after G₀, and differentiation. We found that myostatin was highly upregulated during proliferation and differentiation while Dlk1 was downregulated, thus an inverse relation was observed. B. Dlk1 (app. 30 kDa product) and myostatin (a double band around 26 kDa, corresponding to the cleavage product) protein level was analyzed with western blotting during proliferation before and after G₀, during G₀ (cell cycle arrest) and differentiation of primary isolated human muscle cells. Myostatin protein was expressed during proliferation and differentiation but down regulated during G₀. Dlk1 protein was predominantly expressed by G₀ arrested myoblasts. Ponceau Red staining (a common 70 kDa band is shown) confirmed equal loading. C: The expression of Dlk1, Myogenin and Desmin was studied in two human rhabodomyosarcoma cell lines, RD and A204 after induction in differentiation medium. We found that both cultures were Dlk1 and Desmin positive, while only RD cells were Myogenin positive. None of the cultures were able to form myotubes when cultured in differentiation medium. Scalebar representative for all images: 20 µm. D. RT-PCR analysis of Dlk1 splice variants present in RMS cells. The primers used span exon 5 and should thus provide bands for all possible splice variants present. The analysis shows the presence of 3 bands in both RMS cell lines and in the positive control tissue used, placenta and human muscle. These bands correspond to the full length A, and two smaller products most likely the B and C/C2 variants. All bands were sequenced and verified to be Dlk1.