Fig. S1. Suppression of tyrosine kinase activity by chemical inhibitors and of Wnt3a-stimulated Lef/Tcf-sensitive transcription by KD of Src family tyrosine kinase Yes. (A) Rfz1-expressing cells were preincubated with or without tyrosine kinase inhibitors for 30 minutes and stimulated with Wnt3a for 8 hours in the presence or absence of inhibitors. Src family tyrosine kinase activity was assayed using Src peptide as a substrate. Phosphate ³²P incorporation into substrate peptide was assayed. The results showed are mean values ± s.e.m. from four independent experiments. (B) F9 cells were treated with siRNA targeting c-Yes 1 day before Rfz1 and M50 co-transfection. Cells were stimulated Wnt3a for 7 hours and Lef/Tcf-sensitive transcription was assayed. Bottom panel shows immunostaining with either anti-c-Yes or anti-GAPDH (as a sample loading control). The results showed are mean values ± s.e.m. from three independent experiments.

Fig. S2. Inhibition of Src TK activity blocks Wnt3a-stimulated Lef/Tcf-sensitive transcription in HEK293 and hMSCs. (A) HEK293 cells were co-transfected with Rfz1 and Super8xTOPFlash (M50) reporter. Prior to a 30 minute Wnt3a stimulation, cells were preincubated in the presence or absence of tyrosine kinases inhibitors, genistein (25 M) or PP2 (1 M) without and with purified Wnt3a for 7 hours. The luciferase gene reporter was assayed and is displayed relative to the unstimulated cells (set to 1).The results shown are mean values ± s.e.m. from six independent experiments (left). F9 cells were treated with siRNAs targeting Src for 1 day before co-transfection of the cells with Rfz1 and M50. Cells were stimulated with or without Wnt3a for 7 hours. The luciferase gene reporter was assayed and displayed relative to the unstimulated cells (set to ‘1’). The results displayed are mean values ± s.e.m. from four separate experiments. Cell lysates were analyzed by immunoblotting; the blots were stained with anti-Src antibody. Immunoblots were stained also with anti-GAPDH antibody to establish loading equivalence. (B) Human mesenchymal stem cells were transfected with M50 by nucleofection. Cells were untreated or pretreated with PP2 (Src TK inhibitor) for 30 minutes and then stimulated with or without Wnt3a for 7 hours in the presence or absence of inhibitor. The Lef/Tcf sensitive transcription was assayed and displayed to the unstimulated cells (set to ‘1’).

Fig. S3. Suppression of Src expression stimulates AP-1 transcription (marker of planar cell polarity pathway), whereas inhibiting Wnt5a-stimuation of NF-AT transcription (marker of non-canonical Wnt5a/Ca²⁺ pathway). (A) Cells were transfected with siRNA targeting Src. Next day, cells were co-transfected with Rfz1 and AP-1 Luc. Cells were treated with or without Wnt3a for 7 hours. AP-1 transcription was assayed. The results are mean values ± s.e.m. from three independent experiments. Bottom panel shows staining with either anti-Src or anti-GAPDH. (B) F9 cells were transfected with Src siRNA. On the following day, cells were co-transfected with Rfz2 and p-NF-AT Luc. Cells were treated with or without Wnt5a for 7 hours. Cell lysates were subjected to luciferase assay. The results are mean values ± s.e.m. from three independent experiments. Bottom panel shows the immunoblots staining with either anti-Src antibody or anti-GAPDH antibody.

Fig. S4. Src overexpression and tyrosine kinase activity: dose-response to transient transfection. (A) Wild-type Flag-tagged-Src was overexpressed by transient transfection of F9 cell at the input levels of vector DNA indicated. Cell lysates were analyzed for activated Src (using antibody against phosphorylated, activated Src, p-Src), exogenously expressed Src (using anti-Flag-tag antibody), total Src (using anti-Src antibody), and equivalency of loading (anti-GAPDH antibody). (B) Src family TK activity was assayed using Src peptide as a substrate. The results are mean values ± s.e.m. from seven independent experiments. Statistical significance is indicated (*P<0.005).

Fig. S5. Wnt3a does not stimulate Dvl2 to dock to Src SH2 domain. Rfz1 expressing cells were stimulated with or without Wnt3a for 30 minutes. Whole-cell lysates then were prepared and incubated with either GST itself (as a control, GST) or immobilized GST fusion Src SH2 domain (SH2) at 4°C overnight. Proteins bound to the matrix were eluted with SDS sample buffer and subjected to SDS-PAGE, immunoblotting, and stained with anti-Dvl2 antibody (top) or anti-GST antibody (bottom). Blots shown are representative of five independent experiments.

Fig. S6. Wnt3a stimulates Src family tyrosine kinase activity: time-course. Rfz1-expressing cells were stimulated with purified Wnt3a for the indicated times (0 to 4 hours). Cells were lysed with RIPA buffer. Src family tyrosine kinase activity was assayed using Src peptide as a substrate. Phosphate ³²P incorporation into substrate peptide was assayed. The results are mean values ± s.e.m. from six independent experiments. Statistical significance is indicated (*P<0.005; **P<0.05).

Fig. S7. Expression of constitutively active Y527F Src enhances Wnt3a-stimulation of Lef/Tcf-sensitive transcriptional response. F9 cells were co-transfected with Rfz1, Super8xTOPFlash reporter, and a Src expression vector harboring either the constitutively active Y527F Src (A) or the kinase-dead Src (K295R mutant, B), 1 day before cells were stimulated without or with purified Wnt3a for 8 hours. Cell lysates were assayed for Lef/Tcf-sensitive transcriptional activity using the Luciferase reported gene. Results are displayed relative to the unstimulated cell (set to ‘1’). The results are shown as mean values ± s.e.m. from six independent experiments. Statistical significance is indicated (*P<0.005; **P<0.05; ***P<0.01). The bottom of panels A and B display cellular content of Src and of GAPDH (as a sample loading control). Representative immunoblots are displayed.

Fig. S8. Src family members (Src and Hck) phosphorylate Dvl2. (A) In vitro phosphorylation of Dv2 by either Src or Hck. Purified rDvl2 was incubated without or with either rSrc or rHck in a phosphorylation reaction buffer containing of γ-³²PATP (500-1000 c.p.m./pmol) at 30°C for 1 hour. The phosphorylation reactions were terminated by addition of SDS-sample buffer and then analyzed by SDS-PAGE and autoradiography. Bands were quantified by a calibrated scanner and results were displayed for a representative experiment (bottom panels). (B) Phosphorylation of Dvl2 domains by Hck. GST fusions proteins engineered with Dvl2 DIX, PDZ, and DEP domains were individually incubated with purified rHck in a phosphorylation reaction buffer for 1 hour. The phosphorylation reactions were terminated and then analyzed by SDS-PAGE and autoradiography (upper panel). Samples were analyzed by SDA-PAGE; immunoblotting with anti-GST antibody was employed to establish loading equivalence (bottom panel).