Figure 4. Mutual exclusion of myogenic differentiation and OTX2 expression.

(A and B) Loss of OTX2 expression by myogenic conversion of D425 cells. GFP-transfected D425 cells were co-cultured with C2C12 mouse myoblasts in differentiation medium. Cells were stained with myogenic marker MYH or desmin (Des) antibody and Texas Red (TX) secondary antibody (A). Expression of OTX2 was visualized by OTX2 antibody and TX secondary antibody (B). Thirty GFP-positive cells with myotube morphology were examined and none of them showed significant OTX2 staining. White arrow heads indicate the multipile nuclei in the myogenic D245 cells, which were stained by DAPI (merge+DAPI). It is worth noting that the polyclonal D425-GFP cells expressed GFP in various levels, which remained OTX2-positive when maintained in the undifferentiated single cell form.

(C) OTX2 knockdown activated myogenic and neuronal differentiation in D425 cells. D425 cells infected with lentivirus of Dox-inducible OTX2-shRNA construct were incubated with 0.1 µg/ml Dox on surface coated with poly-D-lysine for 10 days. Desmin or β-tubulin III staining visualized the cells undergone myogenic or neuronal differentiation. The white arrow head indicates a desmin-positive D425 cell with three nuclei. Bright field pictures with phase contrast were shown next to the immunofluorescent staining. Control D425 cells infected with mock lentivirus did not shown positive staining of desmin and β-tubulin III (data not shown).