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. Author manuscript; available in PMC: 2017 Sep 19.
Published in final edited form as: Cell. 2011 Jun 10;145(6):875–889. doi: 10.1016/j.cell.2011.05.017

Figure 2. Down regulation of ES cell specific factors is necessary for differentiation.

Figure 2

(A) Schematic of ES cell differentiation protocol. 48 hours after the withdrawal of pluripotency promoting conditions (LIF and BMP), cells are exposed to differentiation signals. Cells then respond to ME inducing signals (Wnt3a or CHIR) by activating Brachyury and to NE inducing signals (retinoic acid or endogenous FGF) by activating Sox1. (B) Histogram of fold change of mRNA expression levels in the cell population 48 hours after withdrawal of LIF and BMP as measured by microarray. Table shows fold changes for a set of pluripotency factors as well as for Dnmt3b and Fgf5. (C) The scatter plot (left) of Oct4 vs. Sox2 expression in n > 1000 single cells 48 hours after withdrawal of pluripotency conditions. The red dot indicates mean expression level of these proteins in pluripotency conditions. Fluorescent images of cells immunostained for DAPI, Tbx3 (above) and Klf4 (below) 48 hours after withdrawal of pluripotency conditions. Scale bar 32μm (D) Scatter plot of Nanog vs. Dnmt3a levels in single cells under pluripotency supporting conditions (black) and 48 hours after the withdrawal of pluripotency conditions (orange). Images of cells immunostained for DAPI, Dnmt3a (red) and Nanog (green) 48 hours after pluripotency condition withdrawal. Scale bars 32 μm. (E) Plot of the fold changes of mean Nanog protein levels (> 20,000 cells) in cell populations fixed every 6 hours following withdrawal of LIF and BMP. Error bars indicate ±SD in Nanog protein levels in the cell population. Solid line shows Nanog decay fit to an exponential with a half-life of 7.5 hours. (F) Fluorescence images of cells in pluripotency conditions, LIF and BMP, stained for DAPI, Nanog, and Brachyury following CHIR addition for 24 hours and siRNA knock-down of Nanog. siRNA construct was added to cells for 24 hours prior to CHIR exposure. Scale bars 32μm.