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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Version of Record online: 1 SEP 2009
Copyright © 2009 AlphaMed Press
Volume 27, Issue 12, pages 2917–2927, December 2009
How to Cite
C̆ajánek, L., Ribeiro, D., Liste, I., Parish, C. L., Bryja, V. and Arenas, E. (2009), Wnt/β-Catenin Signaling Blockade Promotes Neuronal Induction and Dopaminergic Differentiation in Embryonic Stem Cells. STEM CELLS, 27: 2917–2927. doi: 10.1002/stem.210
Author contributions: LC: conception and design, collection and/or assembly of data, data analyses and interpretation, manuscript writing, final approval of manuscript; DR: collection and/or assembly of data, data analyses and interpretation; IL: collection and/or assembly of data, data analyses and interpretation; CLP: collection and/or assembly of data, data analyses and interpretation; VB: conception and design, data analyses and interpretation, manuscript writing; EA: conception and design, fund raising, data analyses and interpretation, manuscript writing, final approval of manuscript.
First published online in STEM CELLS EXPRESS September 1, 2009.
Disclosure of potential conflicts of interest is found at the end of this article.
- Issue online: 14 DEC 2009
- Version of Record online: 1 SEP 2009
- Accepted manuscript online: 1 SEP 2009 12:00AM EST
- Manuscript Accepted: 19 AUG 2009
- Manuscript Received: 19 MAR 2009
- European Union (Neurostemcell)
- Swedish Foundation for Strategic Research
- Swedish Research Council
- Norwegian Research Council and Karolinska Institute
- Ministry of Education, Youth, and Sports of the Czech Republic. Grant Number: MSM0021622430
- Academy of Sciences of the Czech Republic. Grant Numbers: AVOZ50040507, AVOZ50040702
- EMBO Installation Grant and Czech Science Foundation. Grant Number: 204/09/0498
- Karolinska Institute. Grant Number: 6110/06-225
- Foundation for Science and Technology from the Portuguese Government. Grant Number: SFRH/BD/24585/2005
- Human Frontiers Science Program long-term fellowship
- National Health and Medical Research Council, Australia, CJ Martin fellowship
|STEM_210_sm_suppinfofigure1.tif||12814K||Supplementary figure 1: (A) Genotyping of wt and ko alleles by PCR showed that all ko mESC lines are homozygotes for the gene of interest. (B) Wnt 1 and LRP6 ko mESC lines showed no apparent differences in colony morphology compared to wt. (C) Western blot analyses showed no difference in expression level of Oct3/4 in Wnt 1 and LRP6 ko mESC lines compared to wt.|
|STEM_210_sm_suppinfofigure2.tif||341K||Supplementary figure 2: mRNA expression of Wnt1 (A) and its target genes Axin2 and Brachyury (B) during feeder free differentiation of mESCs. mRNA levels were normalized to their corresponding control conditions: Wnt1 expression in ventral midbrain embryonic day 11.5 (VM E11:5) in A, or in wt mESCs at day 4 of differentiation. Graphs represent summary of at least 4 experiments, * p≤0.05.|
|STEM_210_sm_suppinfofigure3.tif||706K||Supplementary figure 3: Characterization of the effects of Dkk1 on Wnt/β-catenin signaling in a dopaminergic cells (A) and mESCs (B and C). (A) Dkk1 decreased the activation of the Wnt/β-catenin pathway. SN4741 cells were treated with increasing doses (ng/ml) of Wnt3a and/or Dkk1 for 2.5 hours and analyzed for level of ABC (unphosphorated, activated β-catenin) and mobility shift of Dvl3 by western blotting. (B) Dkk1 decreased the activation of the Wnt/β-catenin pathway in mESCs treated with Wnt3a. LRP6 wt and ko mESCs were treated for 2.5 hour with Wnt3a (50 ng/ml), and Dkk1 (100ng/ml or 400 ng/ml) or vehicle, as indicated. (C) wt mESCs were analyzed for LRP5/6 phosphorylation and ABC level by western blot. Cells were treated with either 0.1% BSA in PBS for 16 hours (vehicle), 500 ng/ml Dkk1for 16 hours and 75ng/ml Wnt3a during the last 4 hours (Dkk1 pretreatment), or vehicle for 16 hours plus 75ng/ml of Wnt3a and 500 ng/ml of Dkk1 during the last 4 hours (Dkk1 co-treatment). Note the lower ABC and phosho-LRP5/6 levels in the Dkk1 co-treatment compared to Dkk1 pretreatment.|
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