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. 2015 Jan 8;6(1):e1588. doi: 10.1038/cddis.2014.551

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Copyright © 2015 Macmillan Publishers Limited

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International Licence. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons licence, users will need to obtain permission from the licence holder to reproduce the material. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0

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Knockdown of Rif1 in Smad3−/− ESCs can attenuate cell migration. (a) Image (upper) and histogram (lower) of scratch wound healing of WT ESCs and control shRNA- and Rif1 shRNA-transduced Smad3−/− ESC-differentiated cells at 3, 12 and 24 h after scratch. The data are shown as the mean±S.D. (n=2). (b) Picture of SCID mice with tumors at 4 weeks after WT ESCs, Smad3−/− ESCs and Rif1 shRNA-transduced Smad3−/− ESCs were subcutaneously injected into SCID mice. (c) Genotyping of ES cell formed tumors to confirm Rif1 shRNA integration in Rif1 shRNA transduced Smad3−/− ES cells. (d) Quantitative real-time PCR to examine the mRNA levels of Smad3, Rif1, Ccnd2, Mmp2 and Mmp9 in tumors grown from WT ESCs, Smad3−/− ESCs and Rif1 shRNA-transduced Smad3−/− ESCs. Actin was analyzed as an internal control. The data are shown as the mean±S.D. (n=3). (e) Immunofluorescence staining with anti-PCNA antibody to examine PCNA expression in WT ESC, control Smad3−/− ESC and Rif1 shRNA-transduced Smad3−/− ESC-formed teratomas. The nuclei were stained with DAPI. Scale bar=200 μm. (f) Quantification of the PCNA-positive cell percentage compared with DAPI in WT ESC, control Smad3−/− ESC and Rif1 shRNA transduced Smad3−/− ESC-formed teratomas. Statistically significant differences, calculated through student's t-tests, are indicated (*P<0.05; **P<0.01; ***P<0.001)