Fig. 4: 5-ASA enhances PPARγ signaling in chondrocytes and chondrogenesis.

a Primary chondrocytes were treated without IL-1β or with 10 ng/ml IL-1β and 10 µM 5-ASA or 10 µM rosiglitazone (Rosi), and Pparg mRNA was measured with qRT-PCR. b Primary chondrocytes were infected with Ad-Control (Ad-C; 800 MOI) or the indicated Ad-PPARγ MOI for 2 h, and Pparg mRNA was measured with qRT-PCR. c Primary chondrocytes were infected with 800 MOI Ad-PPARγ and then treated with 10 ng/ml IL-1β with or without 5 µM 5-ASA. COX-2/Ptgs2 mRNA was determined by qRT-PCR (n = 5 independent primary cultures in a–c). d Articular cartilage from ten patients obtained during total knee replacement surgery was subjected to Alcian-Blue staining (left) and IHC for OSCAR and PPARγ expression (middle and right, respectively). The damaged and undamaged sections were assessed separately. Representative Alcian blue-stained (scale bar, 100 µm) and IHC (scale bar, 50 µm) images are shown. e Murine bone marrow-derived MSCs were cultured in a chondrogenic medium (CM) with or without 5-ASA for 7, 14, and 21 days, and qRT-PCR was conducted to determine Oscar. f–g Bone marrow-derived MSCs were cultured in a chondrogenic medium with or without 5-ASA for 3 weeks, stained with Alcian Blue, and photographed (left, f). Alcian-Blue activity was quantified by measuring the absorbance at 630 nm (right, g). qRT-PCR was conducted to determine Oscar, Col2a1, Acan, Pparg, Sox9, and Rxra mRNA expression (g). h Bone marrow-derived MSCs were subjected to pellet culture in e-tubes for 3 weeks with or without 0.01, 0.1, 1, and 10 µM 5-ASA, after which the pellet diameter was measured with a micro ruler (n = 5 independent primary cultures in e–h). i Human chondrocytes were subjected to spheroid culture in a 3D microenvironment with or without 0.01, 0.1, 1, and 10 µM 5-ASA for 1 week, and the diameter of the spheroids was measured (n = 5 independent cultures in i). The data are shown as means ± s.e.m. P-values were obtained by one-way ANOVA followed by Tukey’s multiple comparisons test (b, c, f–i) or two-way ANOVA followed by Sidak’s post hoc test (a, e). Exact P-values were provided in the Source Data file.