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. 2015 Aug 18;106(10):1351–1361. doi: 10.1111/cas.12746

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© 2015 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

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Embryoid body (EB)-mediated differentiation of SH-IN 4F cells. (a) Representative phase contrast images of EBs generated from SH-IN 4F cells. Undifferentiated cells at day 0 and differentiated EBs at days 2, 7, and 10. Scale bar: 100 μm. (b) Expression of ectodermal (PAX6 and SOX1), mesodermal (HAND1 and FOXF1) and endodermal (AFP and GATA6) markers were examined by qPCR. The x-axis represents each time point in days and the y-axis represents relative fold induction compared with day 0. All values were normalized to β-actin mRNA expression levels. (c) Immunofluorescence analysis of SH-IN 4F (clone 2) after EB differentiation: endoderm (GATA6), mesoderm (α-smooth muscle actin; [α-SMA]) and ectoderm (βIII tubulin). Nuclei were stained with DAPI (blue). Scale bars: 75 μm (GATA6 and α-SMA) and 50 μm (βIII tubulin). Results are representative of three independent experiments.