FIGURE 10.

Modulation of WNT2 signaling in bladder SMCs and its effect on cell size, proliferation, and gene expression. A, overexpression (overex) of WNT2 in bladder SMCs. QPCR was used to determine the mRNA levels of WNT2 in bladder SMCs expressing scrambled (scr) miR control, miR-199a-5p, antimiR-199a-5p, and WNT2 protein. Graphs show the results related to scrambled miR control in three independent experiments ±S.E. (**, p < 0.005). Western blotting with anti-WNT2 polyclonal antibodies detects WNT2 protein in HEK293 cells transfected with WNT2-overexpressing plasmid and in bladder SMCs transduced with WNT2-expressing lentivirus. The position of WNT2 protein is indicated with an arrow. B, WNT2-overexpressing bladder SMCs were imaged live at the same magnification as scrambled miR control- and antimiR-199a-5p-expressing cells. Scale bars, 50 μm. C, cell size of the WNT2- and antimiR-199a-5p-expressing SMCs compared with the scrambled miR control ±S.D. (error bars) (**, p < 0.005). D, scrambled miR-, miR-199a-5p-, and antimiR-transduced SMCs were treated with human recombinant WNT2 (100 ng/ml) and DKK1 (50 ng/ml) for 72 h before examining live using an LSM. Scale bars, 100 μm. E, cell surface area was measured (n = 60 cells). Graphs show the averages ± S.D. (error bars) (*, p < 0.05; **, p < 0.005; ns, not significant). F, cell proliferation following WNT2 treatment was analyzed by a BrdU incorporation assay. Cells were seeded at 2 × 10³ cells/well (n = 6 per measurement), grown for 72 h, and treated with recombinant WNT2 for a further 48 h followed by BrdU labeling and a proliferation assay. The graph shows averages of three measurements. Differences from untreated scrambled miR are indicated (**, p < 0.005). G, mRNA levels of WNT-dependent Axin2 and KLF4 genes were determined by QPCR in untransduced bladder SMCs treated with recombinant WNT2 or DKK1 for 48 h. Average (n = 3) -fold differences from the mRNA levels in untreated SMCs ±S.E. (error bars) are shown (*, p < 0.05).