Protein expression analysis.

n.d. = not determined.Expression of Nanog, Oct4, Sox2, VNP and GFP proteins in Nd, E14tg2a and TNG-A cells, grown in serum/LIF or 2i media, and measured by IF, FC-IS or FC in non-fixed cells. Averages and standard deviations of at least 3 independent experiments are depicted (except for TNG-A cells, where only two biological replicates were performed). Nanog expression is not statistically different between different cell lines grown in the same media, although IF measurements provide slightly lower values than FC-IS, probably due to higher sensitivity of cytometry analysis. ES cells grown in 2i media significantly increase Nanog expression (determined by FC-IS) when compared to serum/LIF conditions for all cell lines. In Nd cells, VNP mimics Nanog expression in both culture conditions. In contrast, the percentage of GFP-positive cells in TNG-A ES cells is much higher than the percentage of Nanog-positive cells in serum/LIF conditions, most likely due to reporter stability. Oct4 and Sox2 are always detected in more than 90% of cells in both media and for all ES cell lines, with no statistically significant differences being observed. See Table S3 for statistical data analysis (p-values).