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Embryonic Stem Cells/induced Pluripotent Stem Cells
Article first published online: 24 APR 2013
Copyright © 2013 AlphaMed Press
Volume 31, Issue 5, pages 953–965, May 2013
How to Cite
Han, C., Gu, H., Wang, J., Lu, W., Mei, Y. and Wu, M. (2013), Regulation of L-Threonine Dehydrogenase in Somatic Cell Reprogramming. STEM CELLS, 31: 953–965. doi: 10.1002/stem.1335
Author contributions: C.H.: conceived the project and analyzed the data; performed all the experiments with the help of J.W. and W.L.; H.G.: performed all the experiments with the help of J.W. and W.L.; Y.M. and M.W.: conceived the project and analyzed the data; wrote the manuscript with the help of C.H. All the authors discussed the results and commented on the manuscript. C.H. and H.G. contributed equally to this work.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS January 25, 2013.
- Issue published online: 24 APR 2013
- Article first published online: 24 APR 2013
- Accepted manuscript online: 25 JAN 2013 07:19AM EST
- Manuscript Accepted: 21 DEC 2012
- Manuscript Received: 27 SEP 2012
- Chinese Academy of Sciences. Grant Number: XDA01020104
- Ministry of Science and Technology of China. Grant Numbers: 2010CB912804, 2011CB966302
- National Natural Science Foundation of China. Grant Number: 31030046
- Fundamental Research Funds for Central Universities. Grant Number: WK2060190018
Additional Supporting Information may be found in the online version of this article.
|sc-12-0909_sm_SupplFigure1.tif||335K||Figure S1. The efficiency of shRNA-mediated knockdown of TDH was confirmed in R1 ES cells by Western blot analysis (A), and by real-time RT-PCR analysis (B).|
|sc-12-0909_sm_SupplFigure2.tif||238K||Figure S2. Oct4-EGFP MEF cells were transduced with either OSCK or OSCK plus TDH. The cells were cultured in the indicated culture medium. 16 days after transduction, GFP-positive colonies were counted. Data were represented as mean±SD from three independent experiments. ns indicates no significant relationship.|
|sc-12-0909_sm_SupplFigure3.tif||564K||Figure S3. (A) Listed were the four miRNA candidates that could target to 3′UTR of TDH. (B) miR-9, miR-149, miR-218 and miR-31 were individually transfected into MEF cells together with the indicated pSICHECK2-based luciferase reporter construct. 24 h after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity (mean±SD). * and ns indicate P<0.05 and no significance, respectively. (C) miR-9, miR-149, miR-218 and miR-31 mimics were individually transfected into R1 ES cells. 36 h after transfection, cell lysates were subjected to Western blot analysis with anti-TDH antibody. (D) The successful expression of miR-9, miR-149, miR-218 and miR-31 was confirmed by real-time RT-PCR analysis.|
|sc-12-0909_sm_SupplFigure4.tif||232K||Figure S4. miR-9 mimics or miR-9 inhibitors were transfected into MEF cells together with the indicated reporter construct as indicated. 24 h after transfection, reporter activity was measured and plotted after normalizing with respect to Renilla luciferase activity (mean±SD). *** indicates P<0.001.|
|sc-12-0909_sm_SupplFigure5.pdf||959K||Figure S5. Structural determinants of the TDH-PRMT5 interaction. (A) Schematic representation of PRMT5 and its mutants used in this study. (B) Wild type PRMT5 and the indicated PRMT5 mutants were individually transfected into HEK 293T cells together with GFP-TDH. 24 h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody. (C) Schematic representation of PRMT5 and PRMT5_. (D) Flag-PRMT5 or Flag-PRMT5_ was transfected into HEK 293T cells together with GFP-TDH. 24 h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody. (E) Schematic representation of TDH and its deletion mutants used in this study. (F) Wild type TDH and the indicated TDH mutants were individually transfected into HEK 293T cells together with Flag-PRMT5. 24 h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody.|
|sc-12-0909_sm_SupplTable1.tif||1089K||Supplementary Table 1|
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