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	Figure 1. IQGAP associates with DVL. show more (A) Interaction between ectopically-expressed hIQGAP1 and hDVL1 in HEK 293 cells. Immunoprecipitates (IP) obtained using anti-FLAG antibody were subjected to Western blotting (WB) with the indicated antibodies. +, present; -, absent. (B) Interaction between endogenous hIQGAP1 and hDVL1 in HEK 293T cells. The cultured cells were stimulated with recombinant human Wnt3A for 6 hours (right panels). (C) Interaction between ectopically-expressed hIQGAP1, hIQGAP2, hIQGAP3 and hDVL isoforms in HEK 293T cells. (C) hIQGAP1. (D) hIQGAP2. (E) hIQGAP3. (F) A schematic of the domains of hIQGAP1 and truncated constructs. (G) Interactions between ectopically-expressed hDVL1 and truncated hIQGAP1 constructs. (H) Interactions among ectopically-expressed hIQGAP1 and truncated hDVL1 constructs. (I) A schematic of the domains of hDVL1 and truncated constructs. doi:10.1371/journal.pone.0060865.g001
	Figure 2. Localization of xDVL2-GFP. (a,b) Localization of xDVL2-GFP in Xenopus animal cap cells at stage 10. (A) xDvl2-GFP localized in punctate structures in the cytoplasm (left). Over-expression of Xfz7 recruited xDVL2-GFP to the plasma membrane (center). xIQGAP1-MO did not affect the membrane localization of xDVL2-GFP induced by Xfz7 (right). (B) Nuclear localization of GFP and xDVL2-GFP caused by over-expression of Xwnt-8 and xIQGAP1-MO. GFP signals (left). DAPI staining of animal cap cells (center). Merge (right). (C, D) The ratio of cells that had nuclear fluorescence signals. The average of ratio was taken with six explants in 3 independent experiments (See Materials and Methods). Error bars represent standard deviation of the mean with six explants. Statistical significance was determined by Student's t-test. (C) The ratio of GFP localized in the nucleus in cells. Xwnt-8 was co-injected in lanes 2. Lane 1: n = 291, 18.2%, lane 2: n = 320, 17.2%. P>0.1 [between lane 1 and lane 2]. (D) The ratio of xDVL2-GFP localized in the nucleus in cells injected with xIQGAP1-MO. Xwnt-8 was co-injected in lanes 3 and 4. Lane 1: n = 918, 26.0%, lane 2: n = 1694, 11.0%, lane 3: n = 263, 46.4%, lane 4: n = 477, 29.4%. P<0.01 [between lane 1 and lane 2], P<0.01 [between lane 3 and lane 4]. (E) Cytoplasmic and nuclear distribution of xDVL2, xIQGAP1 and catenin in animal cap cells. MYC-tagged xDVL2 mRNA (100 pg) was injected into the animal poles of 4-cell stage embryos, and the injected animal caps were dissected at stage 10. Lysates from the animal caps were fractionated and subjected to Western blotting with indicated antibodies. Each relative intensity was measured by ImageJ, and its relative ratio was calculated against Input with beta-tubulin for cytoplasm or with Histone H3 for nuclear. Error bars represent standard deviation of the mean in three experiments. Statistical significance was determined by Student's t-test. P<0.1 [between lane 5 and lane 6], P<0.1 [between lane 7 and lane 8]. doi:10.1371/journal.pone.0060865.g002
	Figure 3. Localization of xIQGAP1-GFP. (A) Nuclear localization of xIQGAP1-GFP in stage 10 Xenopus animal cap cells over-expressing Xwnt-8 and xDVL1, 2, 3-MO. GFP signals (left). DAPI staining of animal cap cells (center). Merge (right). (B, C)The ratio of cells that had nuclear fluorescence signals. The following procedure is indicated in Figure 2C. (B) The ratio of nuclear localized xIQGAP1-GFP in cells injected with xDVL2-MO. Lane 1: n = 388, 22.7%, lane 2: n = 361, 22.2%, lane 3: n = 616, 20.9%, lane 4: n = 534, 39.7%. P>0.1 [between lane 1 and lane 2], P<0.05 [between lane 3 and lane 4]. (C) The ratio of nuclear-localized xIQGAP1-GFP in cells injected with xDVL1-MO, xDVL2-MO and xDVL3-MO in Xenopus animal cap cells at stage 10. Lane 1: n = 1424, 23.0%, lane 2: n = 1306, 21.6%, lane 3: n = 1702, 37.6%, lane 4: n = 1409, 16.6%. P>0.1 [between lane 1 and lane 2], P<0.01 [between lane 3 and lane 4]. (D) Cytoplasmic and nuclear distribution of xIQGAP1 in animal cap cells. MYC-tagged xIQGAP1 mRNA (100 pg) was injected into the animal poles of 4-cell stage embryos. The following procedure is indicated in Figure 2C. P<0.1 [between lane 5 and lane 6], P<0.1 [between lane 7 and lane 8]. (E) Interaction between ectopically-expressed xDVL2 and xIQGAP1 in HEK 293T cells. The transfected cultured cells were stimulated with recombinant human Wnt3A for 6 hours in lanes 3 and 4. The bars represent the IP/Input ratios of xDVL2-FLAG for each transfection. Error bars represent standard deviation of the mean in three experiments. Statistical significance was determined by Student's t-test. P<0.1 [between lane 2 and lane 4]. doi:10.1371/journal.pone.0060865.g003
	Figure 4. xIQGAP1 is required for the canonical Wnt pathway during early embryogenesis. (A) Quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). Each indicated morpholino (15 ng) was injected into two dorsal blastomeres of 4-cell embryos. RNAs from dissected dorsal sectors of injected embryos were extracted at stage 10. RNAs from dissected dorsal and ventral sectors of uninjected embryos were used as controls. The value obtained for each gene was normalized to the level of ODC (ornithine decarboxylase). The value of dorsal sectors was set to 100 and other values were computed. Error bars represent standard deviation of the mean in three experiments. Statistical significance was determined by Student's t-test for each marker gene. The highest P values in three marker genes were chosen as a representative, as follows: P<0.05 [between lane 3 and lane 4], P<0.05 [between lane 3 and lane 5], P>0.1 [between lane 3 and lane 6]. (B) Phenotypes of injected embryos at stage 30. Control (upper panel). Xwnt-8 mRNA (0.5 pg) was co-injected with xIQGAP1-, xIQGAP2- or xIQGAP3-MO (15 ng) into two ventral blastomeres of 4-cell embryos. (C) The ratio of injected embryos exhibiting a partial secondary axis. (D) Quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). xIQGAP1-, xIQGAP2- or xIQGAP3-MO (15 ng) and Xwnt-8 (0.5 pg) (20 pg) mRNA were ventrally co-injected. RNAs from dissected ventral sectors of injected embryos were extracted at stage 10. The following procedure is indicated in Figure 4A. P<0.01 [between lane 3 and lane 4], P<0.1 [between lane 3 and lane 5], P>0.1 [between lane 3 and lane 6]. (E) Quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). xIQGAP1, xIQGAP2 or xIQGAP3 (400 pg) mRNA was dorsally injected. The following procedure is indicated in Figure 4A. P<0.05 [between lane 1 and lane 3], P<0.01 [between lane 1 and lane 4], P>0.1 [between lane 1 and lane 5]. (F) Quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). xIQGAP1-MO (15 ng) and Xwnt-8 (0.5 pg) (20 pg) mRNA were ventrally co-injected with xIQGAP1 constructs: xIQGAP1 (400 pg), xIQGAP1-ΔDBR (400 pg), xIQGAP1-DBR (400 pg) mRNA. RNAs from dissected ventral sectors of injected embryos were extracted at stage 10. The following procedure is indicated in Figure 4A. P<0.05 [between lane 3 and lane 4], P<0.05 [between lane 4 and lane 5], P<0.05 [between lane 5 and lane 6], P<0.05 [between lane 5 and lane 7]. (G) The ratio of injected embryos exhibiting a partial secondary axis. The numbered lanes indicate the injected mRNAs and MOs consistent with the numbering in Figure F. (H) The ratio of injected embryos that exhibited a secondary axis. Control-MO (15 ng) or xIQGAP1-MO (15 ng) was co-injected with Siamois mRNA (indicated dose). doi:10.1371/journal.pone.0060865.g004
	Figure S1. Expression of Xenopus DVL and IQGAP1 isoforms and confirmation of the morpholino specificity. Reverse transcriptionolymerase chain reaction analysis was performed using total RNA extracted from Xenopus embryos at different stages of development and from different regions. Ornithine decarboxylase (ODC) was used as an internal control. (A) Temporal expression patterns. U, unfertilized eggs. The numbers indicate developmental stages. (B) Spatial expression patterns. Embryos were dissected at stage 10, and dissections were performed as shown in the right panel. D, dorsal; Vn, ventral; A, animal; M, marginal; Vg, vegetal; H, head. (C) Morpholino (MO) (10 ng) and FLAG-tagged mRNAs (100 pg) were co-injected with globin-FLAG mRNA (100 pg) as loading control into the animal poles of 4-cell stage embryos, and the injected animal caps were dissected at stage 10. Lysates from the animal caps were subjected to Western blotting with anti-FLAG antibody (M2, Sigma). doi:10.1371/journal.pone.0060865.s001
	Figure S2. Localization of xDVL2 and xIQGAP1 GFP constructs in Xenopus animal cap cells at stage 10. (A) GFP signals (left panels). DAPI staining (center panels). Merge (right panels). xDVL2-ΔIBR-GFP (upper panels). xDVL2-IBR-GFP (second panels). xIQGAP1-ΔDBR-GFP (third panels). xIQGAP1-DBR-GFP (bottom panels). (B-E) The ratio of cells that had nuclear fluorescence signals. The average of ratio was taken with six explants in 3 independent experiments (See Materials and Methods). Error bars represent standard deviation of the mean with six explants. Statistical significance was determined by Student's t-test. (B) The ratio of nuclear-localized xDVL2-ΔIBR-GFP. Lane 1: n = 499, 22.4%, lane 2: n = 349, 23.8%. P>0.1 [between lane 1 and lane 2]. (C) The ratio of nuclear-localized xDVL2-IBR-GFP. Lane 1: n = 740, 78.9%, lane 2: n = 420, 87.6%. P>0.1 [between lane 1 and lane 2]. (D) The ratio of nuclear localized xIQGAP1-ΔDBR-GFP. Lane 1: n = 1205, 13.0%, lane 2: n = 410, 14.6%. P>0.1 [between lane 1 and lane 2]. (e) The ratio of nuclear localized xIQGAP1-DBR-GFP. Lane 1: n = 1598, 80.6%, lane 2: n = 408, 92.7%. P<0.01 [between lane 1 and lane 2]. doi:10.1371/journal.pone.0060865.s002
	Figure 5. The role of the xIQGAP1-binding region of xDVL2 in canonical Wnt signaling during early embryogenesis.(A) Quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). Control-MO (15 ng) or xDVL2-MO (15 ng) was ventrally co-injected with Xwnt-8 (0.5 pg) mRNA. RNAs from dissected ventral sectors of injected embryos were extracted at stage 10. The following procedure is indicated in Figure 4A. Error bars represent standard deviation of the mean in three experiments. Statistical significance was determined by Student's t-test for each marker gene. The highest P values in three marker genes were chosen as a representative, as follows: P>0.1 [between lane 3 and lane 4]. (B) Quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). xDVL1-MO (10 ng), xDVL2-MO (10 ng), xDVL3-MO (10 ng) and Xwnt-8 (0.5 pg) mRNA were ventrally co-injected with xDVL2 constructs: xDVL2 (25 pg), xDVL2-ΔIBR (25 pg), xDVL2-IBR (25 pg) mRNA. RNAs from dissected ventral sectors of injected embryos were extracted at stage 10. The following procedure is indicated in Figure 4A. P<0.05 [between lane 3 and lane 4], P<0.05 [between lane 4 and lane 5], P<0.1 [between lane 5 and lane 6], P<0.05 [between lane 5 and lane 7]. (C) The ratio of injected embryos that exhibited a partial secondary axis. The numbered lanes indicate the injected mRNAs and MOs consistent with the numbering in Figure B. (D) Quantitative RT-PCR analysis of early dorsal Wnt target genes (n = 3). Xwnt-8 (0.5 pg) mRNA was ventrally co-injected with xIQGAP1 constructs: xIQGAP1 (400 pg), xIQGAP1-ΔDBR (1 ng), xIQGAP1-DBR (1 ng) mRNA. The following procedure is indicated in Figure 4A. P<0.01 [between lane 3 and lane 4], P>0.1 [between lane 3 and lane 5], P<0.05 [between lane 3 and lane 6].

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