Ectopic expression of sFRP1 in MDA-MB-231 breast cancer cells. (a) Western analysis was performed on lysates of three MDA-MB-231/secreted Frizzled-related protein 1 (sFRP1) clones and three MDA-MB-231/control clones and the levels of active β-catenin, total β-catenin, α-tubulin and DVL1 to DVL3 determined with specific antisera. Myc-tagged sFRP1 was detected with a Myc-specific antiserum. Lower panel: level of total ERK1/2 and P-ERK1/2 in a MDA-MB-231/sFRP1 clone and a MDA-MB-231/control clone. (b) Activity of the WNT/β-catenin pathway was measured using the TOPFlash T-cell factor reporter system. MDA-MB-231/sFRP1-P1 and control-P1 cells were transiently co-transfected with the TOPFlash reporter plasmid and a pRL-CMV control plasmid, and the reporter activity was measured 48 hours later with a luminometer. y axis, TOPFlash reporter activity normalized by pRL-CMV control activity (arbitrary units), average ± standard error. *P < 0.05. (c) Level of Myc-tagged sFRP1 in two pools of MDA-MB-231/sFRP1 cells determined by western analysis using a Myc-specific antiserum. α-Tubulin levels served as a control. P1 is a mixture of the three clones shown in (a); P2 was generated from > 100 sFRP1-infected clones. (d) MDA-MB-231/sFRP1-P1 and (e) MDA-MB-231/sFRP1-P2 and control-P1 and control-P2 were seeded on six-well dishes (200,000 cells/well) in DMEM, 10% FCS. After 1 day and 3 days, three wells per condition were counted and the average cell numbers were calculated ± standard error of the mean. **P < 0.01, *P < 0.05, n.s. = not significant.