Figure 2.

Recombinant ECM1 protein negatively affects chondrocyte differentiation. A) Characterization of recombinant ECM1a protein. Purified recombinant human ECM1a (10 ng/μl in 0.2% bovine serum albumin) was detected by Western blotting with a polyclonal anti-ECM1 antibody. B) Effect of ECM1 conditioned medium on expression of molecules that control chondrocyte maturation assayed by real-time PCR. Pellet culture of hMSCs in either control or ECM1-conditioned medium for 7 d, and real-time PCR was performed. Experiments were carried out in biologic replicates (n = 3). C–H) Recombinant ECM1a protein regulated the expression of genes critical for chondrogenesis, including Sox5 (C), Sox9 (D), Col II (E), Col X (F), PTHrP (G), and IHH (H), assayed by real-time PCR. Micromass cultures of C3H10T1/2 cells were stimulated with 300 ng/ml BMP-2 protein for various time points as indicated in presence or absence of ECM1, and levels of mRNAs were determined by using real-time PCR. Experiments were carried out in biologic replicates (n = 3). Units are arbitrary and normalized values were calibrated against controls, here given the value of 1. Rel. level, relative level. Values are means ± sd. *P < 0.05 vs. PBS treatment control group.