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sc-11-1129_sm_SupplFigure1.pdf919KSupplementary Figure 1. Radioactive in situ hybridization for ShcD in E10.5, E12.5 and E16.5 mouse embryos. The upper row shows sagittal sections and the lower row shows transverse sections of the embryos, at different stages. Regions of signal are indicated by abbreviations and/or white arrowhead. In summary, ShcD transcripts were evidenced in the dorsal root ganglia, cranial ganglia and regions of the developing brain. Abbreviations: anterior hypothalamus (ahy), basal ganglia (ba), cephalic ganglion V (V), cephalic ganglion IX (IX), cerebellum (cb), cerebellar precursors (cbp), cortex (cx), primordial cochlea (co), dorsal aorta (da), diencephalon (di), dorsal root ganglia (drg), dorsal thalamus (dt), forebrain (fb), intestines (gu), internal ear (ie), kidney (ki), liver (li), lungs (lu), mandible (ma), midbrain (mb), mesencephalon (ms), olfactory bulb (ob), olfactory epithelium (oe), optic vesicle (ov), pineal gland (pi), pons (po), rostral migratory stream (rms), septum of forebrain (se), telencephalon (te), tongue (to).
sc-11-1129_sm_SupplFigure2.pdf938KSupplementary Figure 2. Characterization of wild-type and derived ShcD+/− and −/− ESCs. (A) Wild-type, ShcD+/− and −/− clone 1 (cl.1) and clone 2 (cl.2) lines that were employed for the studies were characterized for embryonic stem cell (ESCs) markers. Panels from left to right: bright field picture of ESCs grown on inactivated feeder cells; alkaline phosphatase staining; immunofluorescent images of Oct4 and merged with DAPI; immunofluorescent images of Nanog merged with DAPI. Scale bar represents 50 μm. Karyotype analysis show normal karyotype of ESCs as reverted images of DAPI. Red dots indicate chromosomes and the total number of chromosomes (2n=40) is indicated in red at the top left corner of each image. (B) qRT-PCR for pluripotency markers, Rex1, Klf4, Fgf4 and Sox2 on wild-type and derived ShcD+/− and −/− lines, grown under self-renewal conditions in ESC medium. mRNA levels of the markers are shown relative to TATA Binding protein (Tbp) levels for each sample. Bars represent the means ± S.D. of triplicates. (C) Western blotting for ShcD in the ESC lines before and after EpiSC differentiation. Ponceaus S staining is shown as the loading control for the blot. (D) Summary of cell cycle phase distribution of the ESC lines analyzed by FACS with propidium iodide (PI) staining. Cells were grown under self-renewal conditions. Bars represent the means ± S.D. of triplicates. (E) Analysis of apoptosis within the ESC cultures under standard selfrenewal conditions by FACS analysis of the sub-G1 population by PI staining. Bars represent the means ± S.D. of triplicates.
sc-11-1129_sm_SupplFigure3.tif2191KSupplementary Figure 3. Multiparameter FACS analysis of cleaved Caspase-3 expression, Oct4 expression and cell proliferation in wild-type ESCs during their differentiation to EpiSCs. Wild-type ESCs were differentiated to EpiSCs and the cultures were analyzed for cleaved Caspase-3, Oct4, ethynyl-2′deoxyuridine (EdU) and propidium iodide (PI) by FACS, at several stages of the differentiation: ESCs, early passage (p2), intermediate passages (p3 and p4) and late passages (p5 and p6). (A) Dotplots of cleaved Caspase-3 staining (columns two and four are dot plots with FITC intensities on the y-axis and PI on the x-axis) during the differentiation. (B) Cleaved Caspase-3 negative cells were analyzed for Oct4 expression. Shown are the dot plots with A647 intensities on the y-axis and PI on the x-axis. Line divides the mean intensity below which cells are considered to be negative for Oct4. (C) The proliferative ability of wild-type ESCs undergoing EpiSC differentiation was assessed by analyzing EdU incorporation together with PI staining, on the viable population (cleaved Caspase-3 negative) and separately for the Oct4-positive and negative population. Shown are dot plots with Pacific Blue intensities on the y-axis and PI on the x-axis. The top row shows dotplots for Oct4-positive population and the bottom row for Oct4-negative population. Shown here are the results of a representative FACS experiment as the timing of the onset of these described phenomena could vary slightly between experiments.
sc-11-1129_sm_SupplFigure4.tif2977KSupplementary Figure 4. Analysis of pluripotency and lineage markers during ESC to EpiSC differentiation. qRT-PCR profiles of pluripotency and early lineage genes on wild-type, ShcD+/− and −/− ESCs during EpiSC differentiation. mRNA levels of the markers are shown relative to Tbp levels for each sample. Bars represent the means ± S.D. of triplicates.
sc-11-1129_sm_SupplFigure5.pdf1415KSupplementary Figure 5. Analysis of extraembryonic markers during ESC to EpiSC differentiation. (A) qRT-PCR for Elf5, Eomes and Gata3 in ShcD+/− and −/− cells at intermediate passage (p4) of differentiation. The relative expression levels for these markers have been normalized to Tbp and bars represent the means ± S.D. of triplicates. (B) qRT-PCR for Hand1 in wild-type, ShcD+/− and −/− cells during ESC to EpiSC differentiation. Its relative expression level was normalized to Tbp and bars represent the means ± S.D. of triplicates.
sc-11-1129_sm_SupplFigure6.pdf915KSupplementary Figure 6. Multiparameter FACS analysis of cell proliferation in Oct4 positive and negative cells by EdU incorporation during ESC to EpiSC differentiation. In order to assess the proliferative capacities of the Oct4-positive and negative cells, the two populations were analyzed separately for their EdU incorporation. Oct4-positive ShcD+/− cells (columns one and two from the left) and Oct4-negative ShcD+/− cells (columns three and four from the left) analyzed for EdU in ESCs, two (p2), three (p3) and four (p4) of differentiation. Columns one and three show the cell cycle of viable cells analyzed (Caspase-3 negative). Columns two and four are dot plots with Pacific Blue intensities on the y-axis and PI on the x-axis. The cell cycle phases were gated according to their EdU positivity and negativity, and DNA content based on their PI intensity. In ESCs, the majority of the cells expressing Oct4 are cycling cells while the majority of the Oct4-negative cells were EdUnegative and in the G0/G1 or G2/M phases. Upon differentiation, both the Oct4- positive and negative populations contain cycling cells. In the ShcD−/− cultures there was a lower proportion of cells that have incoporated EdU in both the Oct4-positive and negative populations.
sc-11-1129_sm_SupplFigure7.pdf414KSupplementary Figure 7. In the absence of ShcD, Cdx2-positive cells and overall activation of pErk1/2 are higher. (A) Single immunostaining for Cdx2 on ShcD+/− and −/− cultures during an intermediate passage (p4) of differentiation. Top row shows the merge of Cdx2 and Dapi images taken at 40X and assembled with mosaic approach. Bottom row shows zoom-in images of the inset area from the corresponding top image. The ShcD−/− population contains a majority of cells growing in isolation and expressing Cdx2 (arrowheads). In the ShcD+/− culture, most cells are growing in tight colonies that are negative for Cdx2. A few cells Cdx2- positive cells are present also in the ShcD+/− culture, occurring at the border of the colonies (arrowhead) or growing in isolation. (B) Single immunostaining for pErk1/2 on ShcD+/− and −/− cultures during an intermediate passage (p4) of differentiation. Top row shows the merge of pErk1/2 and Dapi images taken at 40X and assembled with mosaic approach. Bottom row shows zoom-in images of the inset area from the corresponding top image. Cells occurring at the border of the colonies or growing in isolation express high levels of pErk1/2 (arrowheads). The majority of the cells in the ShcD−/− population showed higher activation of pErk1/2.
sc-11-1129_sm_SupplFigure8.tif2808KSupplementary Figure 8. At a late passage of ESC to EpiSC differentiation, ShcD+/− and −/− cultures have overall similar levels of pErk1/2. High-content immunofluorescence analysis for Oct4, Cdx2, pErk1/2, EdU and DAPI on late passage (p6) of differentiation of ShcD+/− and −/− ESCs. For each genotype, images taken at 40X and assembled with mosaic approach are shown. At p6 Cdx2 cells are scarse also in the −/− cultures and the overall activation levels of pErk1/2 are similar.
sc-11-1129_sm_SupplMovie1.wmv3766KSupplementary Movie 1. Time-lapse movie of Oct4-GFP ESCs grown under standard culture conditions imaged by wide-field microscopy with 20X magnification using fluorescence optics. Images were taken every 30 mins for 24 h. The migratory paths of observed cells are traced with a line and the current position with a dot. A cell expressing low levels of GFP (marked in blue) loses GFP expression completely. However, it retains the capacity to proliferate and undergo cell division, giving rise to another GFP-negative cell (marked in green).
sc-11-1129_sm_SupplMovie2.wmv3729KSupplementary Movie 2. Time-lapse movie of Oct4-GFP ESCs grown under standard culture conditions imaged by wide-field microscopy with 20X magnification using fluorescence optics. Images were taken every 30 mins for 24 h. The migratory paths of observed cells are traced with a line and the current position with a dot. Three different behaviours with respect to GFP expression can be observed from this movie: Cells that maintain GFP positivity (marked in pink), cells that are initially GFP-positive and become negative (marked in red then in cyan), and cells negative for GFP that remain negative (marked in blue and green).
sc-11-1129_sm_SupplMovie3.wmv3738KSupplementary Movie 3. Time-lapse movie of Oct4-GFP ESCs grown under standard culture conditions imaged by wide-field microscopy with 20X magnification using fluorescence optics. Images were taken every 30 mins for 24 h. The migratory paths of observed cells are traced with a line and the current position with a dot. GFP-positive and negative cells exhibit distinct morphological and migratory characteristics. An example of a GFPnegative cell (marked in blue) that does not maintain contact with other cells, migrates extensively while gradually increasing its size, in contrast to GFP-positive cells that are smaller in size, grow in tightly packed colonies and show little migration.
sc-11-1129_sm_SupplMovie4.wmv3732KSupplementary Movie 4. Time-lapse movie of Oct4-GFP ESCs grown under standard culture conditions imaged by wide-field microscopy with 20X magnification using fluorescence optics. Images were taken every 30 mins for 24 h. The migratory paths of observed cells are traced with a line and the current position with a dot. A portion of GFP-negative cells undergo endoreduplication giving rise to polyploid cells. The enlarged cell not engaged in colony formation (marked in blue), detaches and reattaches without cell division resulting in a cell containing two nuclei (marked in green).
sc-11-1129_sm_SupplMaterial.pdf26KSupplementary Data

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