NOS network and reporters expression for cell lines grown in serum/LIF conditions.

<p>(A) Immunofluorescence detection for Nanog (red) and VNP/GFP (green) in E14tg2a, Nd and TNG-A cell lines; nuclei counterstained with DAPI (blue). Nanog expression is observed in approximately 50% of cells for all cell lines, while VNP is expressed in the same percentage of Nd cells, with a high degree of co-localization. Higher number of GFP positive cells than Nanog-positive is observed for TNG-A cells. Scale-bar = 50 um. (B) Representative dot blots for FC-IS analysis of Nanog and VNP/GFP for E14tg2a, Nd and TNG-A cells. Negative controls (samples with no primary antibody) for both Nanog and VNP/GFP staining’s are shown, based on which the positive gate regions were designed. Nanog expression is similar between different cell lines grown in the same media. In Nd cells, VNP levels mimic Nanog expression in both culture conditions, while in TNG-A cells, GFP levels are much higher than Nanog levels, most likely due to reporter stability. (C) Representative dot blots for FC-IS analysis of Oct4 and Sox2 for E14tg2a and Nd. Negative controls (samples with no primary antibody) for both Oct4 and Sox2 staining’s are shown, based on which the positive gate regions were designed. Oct4 and Sox2 levels are always expressed in more than 90% of the cells in both media conditions. (D) Correlation plot for Nanog and VNP proteins expression in Nd cells. Graph depicts data from 24906 cells from three biological replicates obtained by FC-IS. Statistical analysis indicates a high degree of correlation between the expression of both proteins (Pearson correlation = 0.647). (E) Representative dot blots for FC analysis of live VNP/GFP for E14tg2a, Nd and TNG-A cells grown. E14tg2a cells were used as negative controls, based on which positive gate regions were designed. Obtained data is similar to that obtained by FC-IS (C). Quantifications of (A–C,E) may be observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059928#pone-0059928-t001" target="_blank">Table 1</a>.