Fig 2: Treatment with rSFRP4 results in decreased Wnt signalling.(A) OVCAR3 cells were co-transfected with pRL-TK (Renilla) and either TOPflash or FOPflash expression plasmids. Cells were subsequently treated with increasing doses of rSFRP4 and/or recombinant Wnt3 a (rWnt3a 0.1 µg/ml) for 48 hours prior to luciferase activities being measured using a Glomax 96 Microplate Luminometer. Average activity and standard deviations were derived from octopulate transfected samples. Results represent the average of 3 experiments and bars represent the standard deviation (s.d) of the mean. *P<0.05. (B) Representative immunoblots showing increased SFRP4 and decreased nuclear ß-catenin protein expression in OVCAR3 cells following 72 hours treatment with rSFRP4 (5 µg/ml). Top panel SFRP4 expression (SFRP4, ab32784, Abcam, Cambridge, UK), second panel a-tubulin expression (a-tubulin #3873, Cell Signalling Technologies, Danvers, MA, USA), third panel nuclear ß-catenin (#sc7963, Santa Cruz Biotechnology), bottom panel Histone H3 (#9715s, Cell Signalling). (C) Densitometric analysis of SFRP4 protein expression in 3 separate experiments. Bars represent the s.d of the mean. *P<0.05. (D) Relative SFRP4 RNA expression was increased in OVCAR3 cells treated with rSFRP4 (5 µg/ml) for 48 hours. qRT-PCR was performed in triplicate and normalised to three different housekeeping genes (SDHA, HSPCB, YWHZA). Results represent an average of 6 experiments. Bars represent the s.d of the mean. *P<0.05. (E) Relative expression of ß-catenin dependent Wnt target genes was decreased in OVCAR3 cells treated with rSFRP4 (5 µg/ml) for 48 hours. qRT-PCR was performed in triplicate and normalised to three different housekeeping genes (SDHA, HSPCB, YWHZA) Expression of AXIN2, MYC and CCND1 were reduced in rSFP4 treated cells, in comparison with the control group. Results represent an average of 4 experiments and bars represent the s.d of the mean. *P<0.05. (F) Relative expression of ß-catenin independent Wnt target genes was unchanged in OVCAR3 cells treated with rSFRP4 (5 µg/ml) for 48 hours. qRT-PCR was performed in triplicate and normalised to three different housekeeping genes (SDHA, HSPCB, YWHZA) Expression of JNK, RHOA, RAC1 and PRK2A were unchanged in rSFP4 treated cells in comparison with the control group. Results represent an average of 3 experiments and bars represent the s.d of the mean.

Fig 3: Addition of rSFRP4 results in MET in ovarian cancer cells.(A) SFRP4 treatment (5 µg/ml, 48 hours) did not cause any obvious changes to the cell morphology of the serous ovarian cancer cell line, OVCAR3 (10× magnification). (B) E-cadherin (CDH1) expression was significantly increased, and Vimentin and Twist decreased in SFRP4 (5 µg/ml, 48 hours) treated OVCAR3 cells. qRT-PCR was performed in triplicate and normalised to three different housekeeping genes (SDHA, HSPCB, YWHZA). Results represent an average of 6 experiments and bars represent the s.d of the mean. **P<0.01, ***P<0.001. (C) Representative immunoblots showing increased CDH1, and decreased VIM and TWIST1 protein expression in OVCAR3 cells following 72 hours treatment with rSFRP4 (5 µg/ml). Top panel CDH1 expression (#3195, Cell Signaling Technology, Danvers, MA, USA), second panel VIM expression(#5741, Cell Signaling Technology), third panel TWIST1 expression (#sc15393, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and bottom panel a-tubulin expression (a-tubulin #3873, Cell Signalling Technology). (D) Densitometric analysis of CDH1, VIM and TWIST1 protein expression in 3 separate experiments. Bars represent the s.d of the mean. **P<0.01, *P<0.05.

Fig 4: Organization of F-actin and microtubules in a cultured western painted turtle hepatocyte. Turtle hepatocytes were isolated as part of another study which was approved by the University of Toronto Animal Care Committee, and turtles were supplied by Niles Biological (Sacramento, CA, USA) and housed as described previously [44]. Hepatocytes were isolated as described by [11] and with sterile conditions, but with the addition of 1X Gibco MEM amino acids, 1X Gibco NEM amino acids, 1X Gibco MEM vitamin solution, 1X Pen-strep (Sigma Aldrich, St. Louis, MO, USA), and 200 µg/mL gentamicin sulphate (Bioshop) to the final media. Cells were cultured for 24 h at 15 °C on glass bottom dishes coated using 2.5 µg/mL purified fibronectin (MilliporeSigma, Burlington, MA, USA) in PBS. Cells were then permeabilized with 0.1% Triton X-100 in PBS for ten minutes, blocked with 7% BSA in PBST with 22.7 mg/mL glycine for one hour, and incubated overnight with mouse anti-a-Tubulin (DM1A) 3873 (Cell Signalling Technology, Danvers, MA, USA) with 1% BSA in PBS overnight at 4 °C. Next, cells were incubated with 1:1000 4',6-diamidino-2-phenylindole DAPI, 1:200 Cy3 conjugated donkey AffiniPure fab fragment anti-mouse (Jackson ImmunoResearch, West Grove, PA, USA) and 1:100 Alexa Fluor 488 Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) in 1% BSA for one hour. The nucleus is presented in blue, tubulin in red, and F-actin in green. Samples were imaged using a Nikon eclipse Ti-2 spinning disk microscope and a Photometrics CoolSnap Myo CCD camera. Data was captured using NIS-Elements Viewer. Scale bar is 10 microns. The sample is focused on the site of attachment to the substrate. F-actin fluorescence staining intensity is observed most abundantly at the cortical cytoskeleton of the cell membrane, especially at adhesions and protrusions, while microtubules are well distributed throughout the cell. The nucleus is not in the focal plane, but its relative position is indicated by out-of-focus fluorescence. Image labels were created with BioRender.com.