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Figure S1 (A) Luciferase reporter assays using the Mix2-290 lux TGFβ reporter in MDA-MB-231 cells treated with TGFβ and transfected with hDvl3 or rFz2. At difference with the pCAGA12 lux, that respond only to Smad3/Smad4, the Mix2-290 promoter is sensitive to Smad2/Smad4. The reporter plasmid pMix2-290lux was always co-transfected with pCS2-myc-FAST1 (2:1 ratio). Data are represented as mean and SD. (B) Overexpression of hDvl3 enhances the induction of the ID1-BRE lux reporter by BMP in HEK293T cells. (C) Luciferase reporter assays using the pCAGA lux TGFβ reporter in MDA-MB-231 cells transfected with the indicated transducers of canonical Wnt signaling, showing no effect. (D) Positive control for the activity of selected transducers of canonical Wnt signaling on the TOP-flash reporter in HEK293T cells transfected with the indicated constructs. Data are represented as mean and SD. (E) Compendium of Fig. 1B; this panel shows that BMP-mediated induction of the ID1-BRE lux reporter is greatly attenuated in cells depleted of Dvl, as obtained by two independent siRNAs. Data are represented as mean and SD. (F) Compendium of Fig. 1B; Loss of Dvl i) limits BMP-mediated induction of the ID1-BRE lux reporter; ii) is rescued by cotransfected (and siRNA insensitive) DvlδDix. Data are represented as mean and SD. (G) Real time PCR analysis with reverse transcription showing relative expression levels of ID2 messenger RNA in MDA-MB-231 cells transfected with Smad4 siRNA and Dvl siRNAs, and treated with BMP2 (100 ng/ml for 2 hours) vs. dorsomorphin (a BMP receptor inhibitor). Induction of ID2 was attenuated upon Dvl depletion. (H and I) In situ hybridization for the markers Xbra and Chordin (Chd) in embryos injected at the 2-4 cell stage with β-catenin morpholino (n=23, 83% and n=15, 100% respectively). (J) Effect of Dvl2/3 depletion on Sizzled expression in embryos injected in the marginal zone radially at the 4 cell-stage with 25 ng of anti-Dvl2 and 25 ng of anti-Dvl3 morpholinos in each blastomere (n=34, 73%). For this experiment we used Dvl MO previously validated in Ref.(4) below. Image published in: Mamidi A et al. (2012) Copyright © 2012 Macmillan Publishers Limited. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives license Gene Synonyms Species Stage(s) Tissue tbxt.S bra, brachyury, ntl, t, t-a, t-b, X-bra, Xbra, Xbrachyury X. laevis Throughout NF stage 10.5 mesoderm marginal zone chrd.S chd, chordin, chrd.1, LOC108716939, X-chordin X. laevis Throughout NF stage 10.5 dorsal marginal zone upper blastopore lip Image source: Published Experiment + Assay Source Phenotypes and Disease Xla Wt + ctnnb1 MO + NF10.5 (in situ hybridization) fig.S1.i Expression Phenotype decreased amount chrd.1.S expression in dorsal marginal zone Xla Wt + dvl2 MO + dvl3 MO + NF10.5 (in situ hybridization) fig.S1.j Expression Phenotype decreased amount szl.L expression in marginal zone Larger Image Printer Friendly View

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