Shoc2 overexpression increases EGF-induced PC12 differentiation.

A), PC12 cells were co-transfected with plasmids pCEFL-KZ-GFP (0.3 µg) and pCEFL-KZ-AU5 (0.7 µg) (top: GFP + EGF) or pCEFL-KZ-GFP (0.3 µg) and pCEFL-KZ-AU5-hShoc2 (0.7 µg) (bottom: Shoc2-GFP + EGF). Transfected PC12 cells were stimulated with EGF (100 ng/ml) for 72 h, fixed, and the presence of extending neurites was analyzed in GFP-positive cells by fluorescence microscopy (right: white arrows). Total cells were visualized with transmitted-light (left). A process equal in length or greater than one cell body was considered a neurite; bars, 10 µm. PC12 cells co-transfected with pCEFL-KZ-GFP and pCEFL-KZ-AU5-hShoc2 but not stimulated with EGF or NGF did not have neurites (not shown). B), PC12 transfected cells (as in A) were stimulated with EGF (100 ng/ml) for 72 h, fixed. Cells were incubated with anti-AU5 monoclonal antibody for 1h at room temperature and, after several washes, were incubated with Alexa 488-conjugated secondary antibodies. The presence of extending neurites was analyzed with transmitted-light (left-picture). PC12 cells overexpressing exogenous Shoc2 were detected as AU5-positive cells by fluorescence microscopy (right-picture and white arrows). A process equal to or greater than one cell body in length was considered a neurite; bars, 10 µm. C), histograms represent the percentage of transfected PC12 cells (GFP-positive: GFP vs Shoc2-GFP) with neurites after EGF treatment of panel A; values are the mean of three separate assays, in which at least 50 GFP-positive cells/assay were analyzed (*P<0.01); bars show SD.