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.