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. 2012 Sep 4;30(11):2423–2436. doi: 10.1002/stem.1217

Figure 1.

Figure 1

Analysis of ShcD expression during ESC differentiation and early embryonic development. (A):ShcD expression analysis by qRT-PCR during neural differentiation of ESCs: ESCs, days 1, 2, 4, 6, and 8 of differentiation and NSCs. ESCs carrying the green fluorescent protein (GFP) reporter under the promoter of the neuroectodermal marker Sox1 (46C) were used to monitor the efficiency of differentiation. ShcD expression level in samples was normalized to the Tbp and ESCs. Data are represented as mean with ± SD. (B): Western blotting for ShcD during neural differentiation of 46C ESCs: ESCs, days 2, 3, 4, 6, and 8 of differentiation. Vinculin is shown as the loading control. (C): qRT-PCR for ShcD in ESCs, EpiSC lines EpiSC1 and EpiSC2 derived from Oct4-GFP mice. mRNA levels of ShcD were determined by normalizing to Tbp and ESCs derived from the same mice. Data are represented as mean with ± SD. (D): Whole mount in situ hybridization of ShcD in early postimplantation embryos at embryonic day E4.5 (peri-implantation), E5.5, E6.5, and E7.5. Whole mount brain preparation of mouse embryo at E10.5 was used as the positive control. (E): Bright-field images of cells for alkaline phosphatase staining of cells before (ESC) and at passage 5 of differentiation (ES-EpiSC). Magnification at ×20. (F): Gene expression profile of EpiSCs at passage 5 of differentiation when EpiSC cultures are established. The relative expression of each gene is compared to its expression level before differentiation. Bars represent the means ± SD of triplicates. (G): qRT-PCR for ShcD during ESC to EpiSC differentiation of wild-type ESCs at different passages: ESC, passages 2, 3, 4, 5, and 10. mRNA levels of ShcD were determined by normalizing to Tbp and ESC. Data are represented as mean with ± SD. (H): Western blotting for ShcD during wild-type ESC differentiation into EpiSCs at progressive passages: from ESCs up to passage p6. Abbreviations: EpiSC1, epiblast stem cell 1; ESC, embryonic stem cell; NSC, neural stem cell; Tbp, TATA binding protein.