Fig. 4. TGFß3 enhances the accumulation of sulfated glycosaminoglycans and COL2 during micromass culture. (A) Cartilage nodule formation in the absence (a-c) and presence (d-f) of TGFß3. The sorted flk-1^–PDGFR⁺ cells (a,d), flk-1⁺PDGFR^– cells (b,e) and DP cells (c,f) were individually spotted in two wells of a 24-well plate at 1.5x10⁵ cells/7.5 µl/spot/well and were subjected to serum-free micromass culture. On day 8, cultures were formalin-fixed and stained with Alcian blue. (B) Higher power view of a-c. Cartilage nodules with a dark blue appearance were noted in the culture of flk-1^–PDGFR⁺ cells (a) and DP cells (c). The Alcian-blue-positive nodules were counted, and the numbers were normalized per 2x10⁵ initial seeding cells and are displayed in the right graph. From three independent experiments, average numbers were calculated and are shown with the corresponding s.d. (vertical line). (C) COL2 accumulation during the micromass culture. (a) The sorted flk-1^–PDGFR⁺ cells (lanes 2,5), DP cells (lanes 3,6), and flk-1⁺PDGFR^– cells (lanes 4,7) were individually spotted in 2 wells of a 24-well plate at 1.5x10⁵ cells/7.5 µl/spot/well, and were subjected to the serum-free micromass culture with 50 ng/ml PDGF-BB (lanes 2-4, P_B) or TGFß3 and 50 ng/ml PDGF-BB (lanes 5-7, TP_B). (b) The sorted flk-1^–PDGFR⁺ cells (lanes 3-5, F–P+), DP cells (lanes 6-8, F+P+), and flk-1⁺PDGFR^– cells (lanes 9-11, F+P–) were also cultured in the same way with TGFß3 (lanes 3,6,9), with TGFß3 and 50 ng/ml PDGF-AA (lanes 4,7,10) or with TGFß3 and 50 ng/ml PDGF-BB (lanes 5,8,11). On day 8, the cultures were harvested with 300 µl/well of loading buffer, and 40 µl aliquots were separated by SDS-PAGE, blotted and developed with 6B3. Soluble COL1 (pepsin-treated) was loaded in lane 1 (b), and soluble COL2 (pepsin-treated) was loaded in lane 1 (a) and lane 2 (b).