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. 2017 Mar 27;7:45311. doi: 10.1038/srep45311

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Copyright © 2017, The Author(s)

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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C9ORF135 is expressed in undifferentiated hESCs and is downregulated during hESC differentiation (a) C9ORF135 expression in undifferentiated ESCs. OCT4 (red), C9ORF135 (green) and merged images in undifferentiated ESCs. (b) C9ORF135 is downregulated during spontaneous differentiation of hESCs. EBs derived from H1 hESCs were digested with Accutase into single cells, seeded on Matrigel-coated glass-bottom dishes and subjected to immunofluorescence analysis. The lineage-specific markers were stained in red and C9ORF135 in green. Nuclei (DAPI, blue). Scale bars, 50 μm. A typical result from triplicate experiments is shown. (c) C9ORF135 mRNA level is downregulated in spontaneous differentiation of hESCs. Nestin and Pax6 are ectoderm markers. KDR and Brachyury T are mesoderm markers. Sox17 and FoxA2 are endoderm markers. Mean values + SD are shown in the figure. (d) C9ORF135 is downregulated during differentiation into three germ layers. EBs derived from H1 hESCs were cultivated in the designated induction medium (phase-contrast images, 4d) in left panels. EBs were digested with Accutase, and the resultant single cells were seeded on Matrigel-coated glass-bottom dishes. Immunofluorescence assay was performed for identifying the pluripotent or specific lineage markers. The lineage-specific markers are labeled in red, and C9ORF135 is shown in green. Nuclei were stained with DAPI (blue). Scale bars, 50 μm. A typical result from triplicate experiments is shown. (e) The C9ORF135 mRNA level is dramatically downregulated during differentiation into three germ layers. The mRNA levels of lineage-specific markers are substantially increased during the differentiation to ectoderm, endoderm or mesoderm cells. The vertical coordinate is a logarithmic scale. Mean values + SD are shown in the figure.

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