Fig. 3.

β-Catenin and Wnt8c cooperate to stimulate chondrocyte maturation. Embryonic chick upper sternal chondrocyte were transfected with the full-length type X collagen reporter (ABC-640-Luc; 100 ng) and lymphocyte enhancer factor 1 (LEF-1) (500 ng) and/or β-Catenin (500 ng) or control vectors after 12 h in culture. Two hours after transfection, standard medium was added and the cultures were harvested 48 h later for luciferase assay. A basic PGL3 vector (10 ng) containing the firefly luciferase gene was used to standardize transfection efficiency. Results are represented as relative luciferase activities obtained by dividing the firefly luciferase by renilla luciferase (A). Similarly, chondrocytes were transfected with ABC-640-Luc (100 ng) or b2-640-Luc (100 ng) and/or β-Catenin (500 ng) and/or T cell factor (TCF)-4 (500 ng) and luciferase assay performed after 48 h (B). Embryonic chick upper sternal chondrocyte were infected with either a control RCAS virus, or viruses containing inserts for either Wnt8c in RCAS BP (B) and/or β-Catenin in RCAS BP (A) (C,D) at the time of plating. After a 48 h culture period in medium containing fresh viral supernatant mixed in a 1:1 ratio with plating medium, fresh control medium was added. Total RNA was extracted from the cultures 3 days later and gene expressions determined by real-time RT-PCR using chick specific primers for either colX (C) or alkaline phosphatase (D). The data shown are representative results of three independent experiments; bars, ±standard error of the mean. The symbol, * represents a significant difference compared to controls with P <0.05.