Fig. 1.

The P2X7 agonist BzATP potentiates β-catenin nuclear localization elicited by Wnt3a. a MC3T3-E1 osteoblast-like cells were seeded at a density of 1.5 × 10⁴ cells/cm² on glass coverslips and cultured for 2 days. Cells were then placed in serum-free media and incubated overnight. The next day, cells were treated under serum-free conditions with recombinant mouse Wnt3a (20 ng/ml) or its vehicle (0.2 % BSA; BSA) in the presence of either BzATP (300 μM) or its vehicle (Vehicle or Veh) and fixed at the indicated times. To observe changes in subcellular localization of β-catenin, immunofluorescence was performed using a β-catenin monoclonal antibody (green) and visualized by confocal microscopy. Data are representative images from four independent preparations each performed in duplicate. Scale bar is 20 μm. b β-catenin nuclear localization was assessed by measuring the average pixel intensity of an area in the nucleus (F _N) and an area of equal size in the cytosol (F _C). The proportion of all cells within 24 fields (12 per coverslip) exhibiting β-catenin nuclear localization was analyzed for each condition. Values of the ratio F _N/F _C greater than or equal to 1.25 were taken as indicating nuclear localization. The number of cells exhibiting nuclear localization was expressed as a percentage of the total number of cells for each treatment group. c The ratio of F _N/F _C (an indication of the intensity of β-catenin nuclear localization) for all cells analyzed was plotted for each treatment at times indicated. For both b and c, α indicates a significant effect of BzATP on Wnt3a-induced β-catenin nuclear localization compared to Wnt3a alone at the same time point (p < 0.05). Data are means ± SEM (n = 4 independent preparations each performed in duplicate)