Fig. 1. Phosphorylation of ß-catenin is inhibited in low-density cultures. (A) Western blot analyses of total cell lysates extracted on days 2 (d2) and 4 (d4) after ES cells were seeded for EB formation at low (LD, 10⁵ cells/ml) and high densities (HD, 10⁶ cells/ml). Density did not alter the levels of total ß-catenin protein. However, the levels of phospho-ß-catenin were significantly reduced in the low-density cultures when compared with the same time point in the high-density cultures using both antibodies (lower two blots). ES cells were seeded for EB formation at either low densities (B) or high densities (C) and stained with anti-ß-catenin antibody. Fluorescence intensity over a random cross-section of an EB demonstrated diffuse staining in low-density EBs (B'), whereas high-density EBs resulted in peaks and valleys in fluorescent intensities (C'). (D) The difference in fluorescence intensity (^*P<0.05, ^**P<0.01) between the peaks and valleys was quantified and plotted graphically. HD EBs had a higher average difference between the peaks and the valleys, demonstrating that ß-catenin staining is more localized (B',C',D, y axis shows fluorescence intensity). (E) Undifferentiated ES cells were transiently transfected with an artificial TCF/LEF promoter and then seeded at high and low densities. Total luciferase activity was then assayed at day 1 post-seeding. High-density EBs repressed basal levels of TCF/LEF activity 2-fold (y axis shows relative luciferase units). (F) ES cells were differentiated by EB formation at low (10⁵ cells/ml) and high (10⁶ cells/ml) densities. On day 4, RT-PCR for Pitx2 was performed in cells treated with RA for 2 hours. Pitx2 is upregulated at low density by RA treatment but not at high density.