Fig 2: Effects of SOD3 on the degradation and synthesis of type I collagen in 3D cultures of fibroblasts. Foreskin fibroblasts embedded in a type I collagen matrix were incubated for 24 h with or without TNF-a (1 ng/mL) and SOD3 (3 µg/mL). The cells in the 3D culture were analyzed via immunofluorescence staining using rabbit anti-type I collagen cleavage-site and Alexa 488-conjugated anti-rabbit IgG antibodies for type I collagen degradation, mouse anti-pN-COL1A1 and Rhodamine Red X-conjugated anti-mouse IgG antibodies for type I collagen synthesis, and Hoechst 33258 for nuclear staining. The cells were analyzed via confocal microscopy. The fluorescence intensities were assessed using the ImageJ software and normalized to nuclear staining. Graphs show the fluorescence intensities for type I collagen degradation and synthesis relative to those in untreated cells. Each value represents the mean ± standard deviation of three independent experiments. * p < 0.05 and ** p < 0.01 vs. control. Magnification, ×100. Bar = 200 µm.

Fig 3: Early and late dedifferentiation biomarkers predict human chondrocyte plasticity. a Confirmation of the immunostained scRNA-seq-defined markers RBP4, SOD3, IFITM3, and F-actin in human P0-8 chondrocytes, as a human chondrocyte dedifferentiation model; scale bars: 50 µm. b Quantitative data of immunostained scRNA-seq-defined markers, cell/nucleus size and cell relative viability in human P0-8 chondrocytes. c Immunostained RBP4, SOD3, IFITM3, and F-actin in human articular chondrocytes from 2 other donors (Donor 1 and 2) for phenotype evaluation; scale bars: 50 µm. d Heatmap of captured parameters in the human chondrocyte dedifferentiation model and articular chondrocyte samples from 2 donors, including immunostained RBP4, SOD3, IFITM3 and F-actin intensity, as well as cell size and nucleus size. Each cell represents the detection of one biomarker in one cell. Each row represents a round of detection. Twenty cells were detected for one biomarker in each batch. e, f Sample-to-sample distance heatmap and principal component analysis (PCA) plot showing the similarity of Donor-1 and Donor-2 chondrocytes with the human chondrocyte dedifferentiation model (P0-8). g Alcian blue–stained micromasses formed by the human chondrocyte dedifferentiation model (P0-8) and Donor-1 and 2 chondrocytes; scale bars: 3 mm. All data are the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Western blot analysis of SOD1, SOD2, SOD3, catalase, and TRX in HUVECs (a–e) and MVs (g). GSH (total content) was analysed using a specific kit (f); the left panel shows the standard curve and the right panel the total GSH content. Y: young; S: senescent. HUVEC protein data were normalized against ß-actin (a–d) or GAPDH (e); however, the GSH data (f) were normalized against the protein content of the sample. Protein data for the MVs was normalized against the intensity of red Ponceau staining. Bars represent mean ± SD (n = 3 pools). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: Effect of recombinant SOD3 treatment on the intracellular ROS levels in fibroblasts. Human foreskin fibroblasts were incubated in a serum-free medium in the presence of TNF-a (1 ng/mL) and/or SOD3 (3 µg/mL) for 24 h. Intracellular ROS levels were measured by incubating the serum-free medium with 10 µM DCF-DA for 30 min and detected as green fluorescence at 525 nm. ROS levels were normalized using crystal violet-stained cell counts. The graph shows ROS levels relative to the levels in untreated cells, which was assessed using the ImageJ software. Each value represents the mean ± standard deviation of three independent experiments. ** p < 0.01 and *** p < 0.001 vs. control. Magnification, ×200. Bar = 100 µm.