Shoc2 overexpression increases EGF-induced PC12 differentiation. Gonzalo Leon Lucia Sanchez-Ruiloba Andrea Perez-Rodriguez Teresa Gragera Natalia Martinez Silvia Hernandez Berta Anta Olga Calero Carlota A. Garcia-Dominguez Lara M. Dura Daniel Peña-Jimenez Judit Castro Natasha Zarich Pilar Sanchez-Gomez Miguel Calero Teresa Iglesias Jose L. Oliva Jose M. Rojas 10.1371/journal.pone.0114837.g003 https://plos.figshare.com/articles/figure/_Shoc2_overexpression_increases_EGF_induced_PC12_differentiation_/1272391 <p><b>A</b>), 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>B</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. <b>C</b>), 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.</p> 2014-12-16 02:49:54 Shoc 2 knockdown egf Shoc 2 protein Shoc 2 overexpression ERK activation Shoc 2 neurite PC 12 cells cell differentiation processes Shoc 2 depletion