Fig 1: Identification of FliI as a potential molecular target involved in OA process(A) Gene enrichment analysis for functional categories of top-regulated genes in murine chondrocytes cocultured with murine macrophages stimulated by cartilage fragments.(B) Violin plots showing the fold change of top-regulated genes in chondrocytes.(C) Expression of FliI in chondrocytes as analyzed by qRT-PCR. Results represent means ± SEM for triplicates and significant difference was determined by the student t-test.(D) Detection of FliI in OA cartilage using IHC. Scale bars are indicated and represent 500 μm for low magnification image and 100 μm for higher magnification images.(E) Detection of FliI in chondrocytes isolated from OA cartilage using IFA. Scale bars represent 50 μm.(F) Detection of FliI in synovial fluids by ELISA (left panel) and Western blotting (right panel).(G) Detection of FliI in supernatant of chondrocyte cultures stimulated with IL-1β or TNF-α using ELISA (upper panel) and Western blotting (lower panel). Results represent means ± SEM for triplicates and significant difference was determined by the One-way ANOVA, followed by Tukey's multiple-comparison procedure.
Fig 2: In vitro stimulation of murine chondrocytes by FliI(A) Dose-dependent manner of FliI in stimulating chondrocytes and promoting the expression of catabolic factors.(B) Gene expression of catabolic factors in chondrocytes stimulated with FliI (5μg/mL) or IL-1β (positive control) as assayed by qRT-PCR. Results represent means ± SEM for 8 samples and significant difference was determined by the one-way ANOVA, followed by Tukey's multiple-comparison procedure.(C) Protein levels of catabolic factors in stimulated chondrocytes as assayed by Western blotting.(D) Gene expression of anabolic factors in stimulated chondrocytes with FliI (5μg/mL) (n = 8). Significant difference was determined by the One-way ANOVA, followed by Tukey's multiple-comparison procedure.(E) Protein levels of hypertrophic factors in stimulated chondrocytes with FliI (5μg/mL) as assayed by Western blotting.(F) Quantification of the relative densities of the detected bands of RUNX2, c-Myc and COL X. Results represent means ± SEM for triplicates and significant difference was determined by the student t-test. Results from in vitro experiments are representative of at least two independent experiments.
Fig 3: Signaling pathway involved in FliI activation(A) FliI activated ERK1/2 in chondrocytes stimulated with FliI (5μg/mL) as evidenced by increased expression of pERK1/2.(B) Dose-dependent manner of FliI in activating ERK1/2 and MEK1/2.(C) Quantification of the relative densities of the detected bands of pERK1/2 and pMEK1/2 for triplicates and significant difference was determined by the One-way ANOVA, followed by Tukey's multiple-comparison procedure.(D) Effects of different doses of MEK1/2 inhibitor U0126 on expression of to ERK1/2, MMP13, MMP3, c-Myc and RUNX2 after FliI activation.(E) Western blot analysis showing that deduction of ERK1/2 expression due to U0126 is associated with reduction of expression of MMP3.(F) Quantification of the relative densities of the detected bands of pERK1/2 and MMP3 for triplicates and significant difference was determined by the One-way ANOVA, followed by Tukey's multiple-comparison procedure.(G) Pull-down assay showing the ability of FliI to bind TLR4 of chondrocytes.(H) Involvement of TLR4 in signaling mediated by FliI activation as analyzed by western blotting.(I) Gene expression of MMP3 and MMP13 in FliI-stimulated chondrocytes after treatment with TAK-242. Results for triplicates and significant difference was determined by the One-way ANOVA, followed by Tukey's multiple-comparison procedure.
Fig 4: Pathological role of FliI in OA process(A) Catabolic effect of FliI on cartilage (Ex-vivo stimulation). Representative images for safranin O staining of sectioned cartilage after Ex-vivo stimulation with FliI (10 μg/mL) or IL-1β. Scale bar represents 50 μm.(B) Quantification of the safranin O staining of sectioned cartilage after stimulation with FliI or IL-1β.(C) Detection of sGAG in supernatant of stimulated cartilage cultures. Results represent means of 9 samples ± SEM and significant difference was determined by the One-way ANOVA, followed by Tukey's multiple-comparison procedure.(D) Experimental design of knockdown experiment.(E) Detection of FliI in transfected chondrocytes.(F) Expression of catabolic factors in naive chondrocytes stimulated by culturing with IL-1β-stimulated-siRNA-transfected chondrocytes. Results represent means ± SEM for 6 samples and significant difference was determined by the student t-test.
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