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. 2009 Jun 1;29(15):4201–4219. doi: 10.1128/MCB.00056-09

FIG. 6.

FIG. 6.

Substrate-binding domain of ADAMTS-7 is important for its regulation of chondrogenesis. (A) Effect of ADAMTS-7 and its C-terminal deletion mutants on chondrocyte differentiation of murine C3H10T1/2 cells. Micromass cultures of control (CTR), wild-type ADAMTS-7 (TS7), or its C-terminal mutants (TS7-M1 to TS7-M6) C3H10T1/2 stable cell lines were stimulated with 300 ng/ml BMP-2 protein for 3 or 7 days, and the mRNA levels of Col II (a), Sox9 (b), Col X (c), Cbfa1 (d), PTHrP (e), and IHH (f) were determined by using real-time PCR. The units are arbitrary, and the normalized values were calibrated against the control (CTR), here given the value of 1. Asterisks indicate a significant increase or decrease from the control (P < 0.05). (B) Effect of ADAMTS-7 and its C-terminal deletion mutants on the expressions of Col II and Col X during chondrogenesis of human MSCs. Human MSCs pellets were cultured for 14 days in the presence of the conditioned medium obtained from the control (CTR), ADAMTS-7 (TS7), or its C-terminal mutants (TS7-M1 to TS7-M6) stably transfected HEK 293-EBNA cell lines, and Col II (a) and Col X (b) expression was analyzed by real-time PCR as described for panel A. (C) Whole-mount alcian blue histochemistry. Staining was performed on the micromass culture of pcDNA3.1 (CTR), wild-type ADAMTS-7 (TS7), and C-terminal four-thrombospondin motifs deletion mutant (TS7-M1) stable lines based on C3H10T1/2 cells treated with BMP-2 protein for 14 days. Rel.level, relative level.