STEM_210_sm_suppinfofigure1.tif12814KSupplementary 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.tif341KSupplementary 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.tif706KSupplementary 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.
STEM_210_sm_suppinfo.doc23KSupporting Information

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