Fig. 3. Induction of chondrogenic markers in CFK-2 cells by Ihh is mimicked by recombinant N-Shh. (A) Alkaline phosphatase (ALP) activity is enhanced by Ihh or N-Ihh, but not by N-Ihh W160G. CFK-2 cells stably transfected with pcDNA3, Ihh or N-Ihh underwent postconfluent growth and were analyzed for ALP enzymatic activity during the indicated time periods (left). ALP activity was also compared in stably transfected CFK-2 cell populations expressing N-Ihh or its mutant variant form, N-Ihh W160G (right). (B) Induction of Col2a1 and Col10a1 collagens by Ihh or N-Ihh. Stably transfected CFK-2 cell populations expressing Ihh, N-Ihh, N-Ihh W160G, or vector alone were subjected to postconfluent growth conditions and total RNA was extracted at the indicated time periods. Northern blot analysis was used to evaluate expression levels of the chondrogenic markers Col2a1 and Col10a1. (C) Dose-response induction of ALP, Col2a1 and Col10a1 by exogenous N-Shh. Naive CFK-2 cells grown to postconfluence were subjected to treatment by vehicle or exogenous N-Shh peptide at the indicated concentrations. After 6 days of treatment, ALP enzymatic activity in cell extracts was measured (left) and total RNA was analyzed for Col2a1 and Col10a1 expression (right). A maximal response was observed at a concentration of 10^-8 M N-Shh.