Fig. 3

ERK2 requires USP15 to influence the TGF-β signaling for regulating the cartilage phenotype in vivo and in vitro. a Simultaneous ERK2 overexpression and USP15 knockout in ATDC5 cells with TGF-β1 (10 ng/mL). b Immunofluorescence staining for Col2a1, Aggrecan, and Col10a1 in ATDC5 cells with or without ERK2 overexpression (scale bars = 50 μm). c The immunofluorescence intensities of Col2a1, Aggrecan, and Col10a1 in each group were calculated, and the data were presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. d–f The expression levels of p-SMAD2, Col2a1, Aggrecan, and MMP13 in the two groups were detected by immunohistochemistry, and the cartilage tissue morphology in the two groups were detected by HE/Safranin-O fast green staining (n = 4 for control groups in the OA models, n = 4 for AAV-mediated USP15 overexpression and ERK2 knockdown groups in the OA models). d Scale bars = 50 μm. e Scale bars = 50 μm. f Scale bars = 200 μm. g The relative expressions of p-SMAD2, Col2a1, Aggrecan, and MMP13 in each group were calculated via immunohistochemistry, and the hyaline cartilage thickness and OARSI scores were quantified. The data were presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. h Relative gene expressions of Col2a1, Aggrecan, and Sox9, which are associated with cartilage anabolic metabolism, were detected by quantitative real-time PCR in two groups (n = 3 for control groups in the OA models followed by real-time quantitative PCR, n = 3 for AAV-mediated USP15 overexpression and ERK2 knockdown groups in the OA models followed by real-time quantitative PCR). The data were presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. All experiments were performed at least three times