Figure 5. GPR17 overexpression inhibits oligodendrocyte differentiation and induces nuclear translocation of ID2/4.

a–b) HCN cells were transfected with GPR17 and control vector carrying GFP and assayed after 72 hrs and immunostained for RIP, MBP and MOG. Arrows indicate the transfected cells. c) Total RNAs extracted from HCN cells transfected with control and GPR17 were subjected to qRT-PCR analysis for Hes5, ID2, ID4 and TCF4. *P<0.01 (Student t-test). d–e) HCN cells transfected with GPR17 (e) or control pCIG (d) carrying nuclear-localized GFP (nls-GFP) were immunostained for ID2. Arrows indicate ID2 immunoreactivity in control- or GPR17-transfected cells, respectively. f) Cytoplasmic (CYTO) and nuclear (NUCL) fractions were prepared 3 days after transfection and subjected to Western blotting analysis for cellular localization of ID2, CREB or GAPDH. g–h) Primary rat OPCs transfected with control or GPR17-expression vector were co-cultured with astrocytes for 5 days and subjected to immunocytochemistry for MBP and GFP. Arrows in g, h indicate MBP+ cells in control- or GPR17-transfected cells. i–j) Cortical progenitors from WT and GPR17-Tg embryos at E15.5 were cultured to promote oligodendrocyte formation. Cells were immunostained with antibodies to O4, MBP and ID2 as indicated. Arrows in j indicate MBP or ID2 expression. k) Cortical progenitor cells from WT, GPR17−/− and CNP1-GPR17 transgenic embryos at E15.5 were cultured to promote oligodendrocyte formation. The cells were collected and processed for co-immunoprecipitation with an antibody against ID2 or ID4. Immunoprecipitated proteins were subjected to Western blot analysis for Olig1 as indicated. Scale bars in a–e, 50 mm; g–h; 25 mm and i–j, 50 mm.