FIG 4.

CypA regulates ATDC5 differentiation through the NF-κB pathway. (A) Relative luciferase activities of NF-κB were determined in WT, SC, 2T7, and 2T3 ATDC5 cells 2 days posttransfection with κB-Luc plasmids. n = 4. *, P < 0.05, and **, P < 0.01 compared to the WT. (B) Western blot analysis of nuclear p65 expression levels. Nuclear extracts prepared from ATDC5 and NF-κB inhibitor-treated ATDC5 cells were subjected to immunoblotting to detect p65 expression levels. p84 was used as a loading control. (C and D) Western blot analyses of ectopic expressed levels of p65 and Sox9 compared to controls. (E to H) The relative mRNA expression levels of Sox9 (E), Col2α1 (F), Col10α1 (G), and Runx2 (H) were determined by qRT-PCR. ATDC5 cells were treated with NF-κB inhibitors at the indicated concentrations and cultured in chondrogenic differentiation medium for 7 days, followed by determination of their relative mRNA expression levels. Cells treated with DMSO were used as controls. n = 4. *, P < 0.05, and **, P < 0.01 compared to the DMSO-treated group. (I to L) Restoration of relative expression levels of Sox9 (I), Col2α1 (J), Col10α1 (K), and Runx2 (L) by transfection of p65 plasmids (p65-p) into CypA Kd ATDC5 2T3 cells. (M to P) Restoration of relative expression levels of Sox9 (M), Col2α1 (N), Col10α1 (O), and Runx2 (P) by transfection of Sox9 plasmids (Sox9-p) into CypA Kd ATDC 2T3 cells. *, P < 0.05 compared to vector-transfected CypA Kd 2T3 cells at the same time points. The data are shown as means and SD of three experiments.