Figure 2
Loss of SFRP1 results in an increase in Wnt-mediated β-catenin activity. A, TERT-pSUPER and TERT-siSFRP1 cells were immunostained with anti-β-catenin and counterstained with DAPI phase contrast images were captured at 20× magnification (scale bar 100 μm) and fluorescent images were captured at 40× magnification (scale bar 50 μm). B, TERT-pSUPER and TERT-siSFRP1 cells were transfected with either the Super8XTOPflash or Super8XFOPflash vectors and relative luciferase activity was measured after an overnight incubation in the presence or absence of the Wnt3a ligand. Bars represent mean ± SEM of relative luciferase activity (firefly luciferase activity/renilla luciferase activity) normalized to the relative luciferase activity in TERT-pSUPER cells treated with control media. C, Twenty-four hours after the cells were treated with either control medium or Wnt3a medium, total RNA was isolated for real-time PCR analysis. The level of Cyclin D1 mRNA was normalized to amplification of GAPDH mRNA, which was performed in parallel wells for each treatment. D, Twenty-four hours after the cells were treated with 3H-Thymidine in either control media or Wnt3a media, the cells were lysed and total cpm was measured. Experiments were performed in at least triplicate wells and bars represent mean ± SEM cpm. *p < 0.05, ***p < 0.001 [significantly different from corresponding TERT-pSUPER cell line using Bonferoni's t-test following two-way ANOVA].