Expression of Ank and generation of extracellular PPi in transduced ATDC5 cells. (a) Extracellular inorganic pyrophosphate (ePPi) levels in various ATDC5 clonal cell lines at day 14 of chondrogenesis. Empty, uncloned ATDC5 cells transduced with pLNCX vector only; WT, wild-type ank. *Significance of ePPi levels of WT and mutant Ank-transduced cell lines versus cells transduced with empty vector only. #Significance of P5L ePPi levels versus cells transduced with WT Ank or mutant ank constructs, as indicated. (b) ePPi levels in various ATDC5 clonal cell lines at day 28 (nonmineralizing hypertrophy) of chondrogenic differentiation. (c) ePPi levels in various ATDC5 clonal cell lines at day 35 (mineralization) of chondrogenic differentiation. WT, P5T, P5L, and M48T are independent clonal cell lines of stably transduced ATDC5 cells exhibiting a 1:1 transcript level ratio of endogenous ank to transduced ank and twice as much Ank protein as untransduced cells. At least three independent clones for each cell line were evaluated; results presented are from single clones and are representative of other clones for each cell line. Inorganic pyrophosphate levels for untransduced ATDC5 cells and empty vector were comparable. n = 9; *P ≤ 0.05. (d) The fold change in the expression of ank mRNA as determined by real-time RT-PCR at various times of chondrogenesis. Black bars, day 14 (proliferation); grey bars, day 28 (nonmineralizing hypertrophy); white bars, day 35 (mineralizing hypertrophy). Note increase of expression in ank under mineralizing conditions, which is consistent with the dramatic increase in ePPi in transduced cells at day 35 of culture.