%0 Figure %A Leon, Gonzalo %A Sanchez-Ruiloba, Lucia %A Perez-Rodriguez, Andrea %A Gragera, Teresa %A Martinez, Natalia %A Hernandez, Silvia %A Anta, Berta %A Calero, Olga %A Garcia-Dominguez, Carlota A. %A M. Dura, Lara %A Peña-Jimenez, Daniel %A Castro, Judit %A Zarich, Natasha %A Sanchez-Gomez, Pilar %A Calero, Miguel %A Iglesias, Teresa %A L. Oliva, Jose %A Rojas, Jose M. %D 2014 %T Shoc2 overexpression increases EGF-induced PC12 differentiation. %U https://plos.figshare.com/articles/figure/_Shoc2_overexpression_increases_EGF_induced_PC12_differentiation_/1272391 %R 10.1371/journal.pone.0114837.g003 %2 https://ndownloader.figshare.com/files/1844840 %K Shoc 2 knockdown %K egf %K Shoc 2 protein %K Shoc 2 overexpression %K ERK activation %K Shoc 2 %K neurite %K PC 12 cells %K cell differentiation processes %K Shoc 2 depletion %X

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.

%I PLOS ONE