Fig. 2. Expression of ß-catenin negatively regulates chondrogenesis in vitro. (A) Mesenchymal cells from chicken embryo were maintained as a micromass culture for 4 days, and distribution of type II collagen and ß-catenin was detected by immunocytochemical staining of cross-sections of a micromass culture spot (top and middle). Subcellular localization of ß-catenin was determined in day-2 culture (bottom). (B) Mesenchymal cells were maintained as micromass culture for the indicated period to induce chondrogenesis (top). Alternatively, the cells were cultured for 4 days in the presence of vehicle alone as a control, 10 nM phorbol ester (PMA), 10 ng/ml epidermal growth factor (EGF), 10 µM SB203580 (SB) or 10 µM PD98059 (PD) (middle and bottom). Type II collagen, ß-catenin and N-cadherin were detected by Western blotting (middle). Synthesis of sulfated proteoglycan was determined by Alcian Blue staining and quantified (bottom). (C) Chondrifying mesenchymal cells during micromass culture were treated with 5 mM NaCl as a control or 5 mM LiCl for indicated periods (top), for 5 days (middle) or for 5 days with the indicated concentrations of LiCl (bottom). Expression of type II collagen, ß-catenin, N-cadherin and phosphorylated GSK-3ß was determined by western blotting (top and middle panels). Accumulation of sulfated proteoglycan at day 4 culture was determined by Alcian Blue staining and quantified (bottom). ERK-2 was detected as loading control of blots.