Fig 2: Celecoxib reduces type X collagen, MMP13 and PTHrP levels and inhibits type II collagen expression in osteoarthritic articular cartilage (A) Immunohistochemical analysis of type X collagen (scale bars: 100 ?µm) (B) MMP13 (scale bars: 200 ?µm) (C) type II collagen, and (D) PTHrP (scale bars: 100 ?µm) in articular cartilage of tibia sections in the OA ?+ ?Saline and OA ?+ ?Celec groups. The histogram illustrates the quantification of the positively stained type X collagen and the relative density of MMP13, type II collagen and PTHrP expression in articular cartilage. Each column represents the mean ?± ?SEM of six samples. Data from each group were compared with those of the OA contralateral control group and were evaluated by one-way ANOVA and Scheffe's test. *, P ?< ?0.05; **, P ?< ?0.01; the OA ?+ ?Celec groups compared to the OA ?+ ?Saline groups #, P ?< ?0.05; ##, P ?< ?0.01.

Fig 3: Inhibition of COX-2 activity by celecoxib and DFU influences downstream targets in cultured HACs (A) Aggrecan (B) Col2a1 (C) Col10a1 (D) PTHrP, and (E) Ihh mRNA expression in vitro in cells treated with celecoxib and DFU (10-6–10-5 ?M) for 7 days. GAPDH mRNA was used as the internal control. Each column represents the mean ?± ?SEM of four replicate cultures. Data from each group were compared with those of the control group and were evaluated by one-way ANOVA and Scheffe's test. *, P ?< ?0.05; **, P ?< ?0.01 compared to the control culture.

Fig 4: COX-2 promotes PTHrP expression by increasing PTHrP promoter activity and suppressing cell hypertrophy (A) Putative COX-2-binding motifs in the PTHrP reporter. The 5'-flanking region of the human PTHrP gene was subcloned into the pGL3 basic reporter. COX-2 activates PTHrP reporter activity in HACs. pcDNA3.1-COX-2 or pcDNA3.1-empty was cotransfected with the PTHrP (-944 to +50) reporter. The data are presented as the mean ?± ?SE of relative luciferase activity from three independent experiments performed in duplicate. **, P ?< ?0.01 compared to the pGL3-empty vector (B) qRT-PCR analysis of PTHrP mRNA expression was performed following transfection with pcDNA3-COX-2 and control pcDNA3-empty vector for 1 and 3 days. GAPDH mRNA was used as the internal control. *, P ?< ?0.05 compared to the control pcDNA3-empty vector (C) Western blot analysis of PTHrP protein levels digitally detected and normalized to GAPDH levels as a loading control (left panel). Each bar represents the mean ?± ?SEM of three replicate cultures (right panel). Data from the pcDNA3.1-COX-2 vector (hCOX2-ov) group were compared with those of the control pcDNA3.1-empty vector (control vector) group and were evaluated by one-way ANOVA. **, P ?< ?0.01 compared to the control vector (D) Flow cytometry evaluating the cell size after transfection of cells with pcDNA3.1-COX-2 and control pcDNA3.1-empty vector for 2 and 3 days. Representative results of the mean cell size are shown in the flow cytometry analysis (left panel). Each column represents the mean ?± ?SEM of six replicate cultures. ***, P ?< ?0.001 compared to the control culture.