Fig 1: The function and expression of TNFAIP3 in ACSCs. a Representative TRAP staining images of osteoclasts during in vitro osteoclast formation in the presence of 5% OASF, 20 ng/mL TNF-α or 250 ng/mL anti-human TNF-α neutralizing antibody showing that TNF-α and OASF (containing TNF-α) suppress osteoclast formation. Scale bar, 50 μm. b Schematic overview of the RNA sequencing workflow. ACSCs were treated with 20 ng/mL TNF-α for 3 d, followed by RNA isolation, cDNA library construction and high-throughput sequencing (n = 3). c GSEA enrichment plots showed that ACSCs were more actively involved in osteoclast-related biological processes after TNF-α stimulation. d Bar plot showing the differential expression of osteoclast-related genes upon TNF-α stimulation. e Venn diagram indicated upregulated osteoclast-related DEGs in TNF-α stimulated ACSCs. f Gene expression of TNFAIP3 in ACSCs treated without or with 20 ng/mL TNF-α. g ELISA quantification of TNFAIP3 in the culture supernatant of ACSCs treated without or with TNF-α (10 ng/mL or 20 ng/mL) for 3 days (n = 3)
Fig 2: ACSC-specific TNFAIP3 overexpression inhibits osteoclastogenesis in vivo. a, b TRAP staining (top) and immunofluorescence of TRAF6 (middle) and CTSK (bottom) (genes associated with OC formation and OC differentiation) in the subchondral bone of OA rats showed that overexpression of TNFAIP3 in ACSCs inhibits osteoclast formation, while knockdown of TNFAIP3 in ACSCs impaired their inhibitory effects in vivo. Scale bars, 200 μm. c Quantification of osteoclast number (left) and the relative fluorescence intensity of TRAF6 (middle) and CTSK (right) in (a–b). The statistical significance of differences was determined using one-way ANOVA with multiple comparison tests (LSD). Experiments were repeated independently for more than three times. All data are shown as the mean ± S.D. **P < 0.01, ***P < 0.001; ns, not significant
Fig 3: ACSC-specific TNFAIP3 overexpression ameliorates surgery-induced OA in rats. a, b 3D reconstruction of rat knee joints (top), tibial subchondral bone medial compartment (sagittal view) (bottom), and microCT images of rat tibia subchondral bone (coronal view) (middle) showed that TNFAIP3-overexpression ACSCs significantly improved the osteochondral structure, while TNFAIP3-knockdown ACSCs lead to poor protection effects in OA rats at 4 week after injection (n = 3). Scale bars, 2 mm. c OA grade was rated by quantification of the degree of cartilage loss and osteophytes from micro-CT. d–f Micro-CT measurements for bone volume fraction (BV/TV) (d), trabecular number (Tb.N) (e) and trabecular separation (Tb.Sp) (f) in the subchondral bone of vector-transfected and TNFAIP3-overexpress or knockdown ACSCs rats at 4 w after injection. g, h Representative images of safranin O–fast green staining (top), toluidine blue staining (middle), and type II collagen immunostaining (bottom) in the subchondral bone showed that TNFAIP3-overexpression ACSCs significantly improved the pathological changes in the osteochondral regions of OA rats, while TNFAIP3-knockdown ACSCs lead to poor effects in OA models at 4 w post-surgery. Scale bars, 200 μm. i Mankin scores of osteoarthritic rat knees at 4 w after injection. The statistical significance of differences was determined using one-way ANOVA with multiple comparison tests (LSD). All data are shown as the mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant
Fig 4: ACSC-derived TNFAIP3 suppresses osteoclastogenesis in vitro. a TRAP staining (top) and immunofluorescence staining of TRAF6 (middle) and CTSK (bottom) showed that the culture supernatant of ACSCs (10%) and soluble TNFAIP3 protein (20 ng/mL) inhibit OC formation and OC differentiation-related gene expression, while addition of the anti-TNFAIP3 neutralizing antibody (100 ng/mL) promotes OC formation. Scale bars, 50 μm. b Quantification of TRAP+ multinucleated osteoclasts (> 3 nuclei) (left) and relative fluorescence intensity of TRAF6 (middle) and CTSK (right) in (a). The statistical significance of differences was determined using one-way ANOVA with multiple comparison tests (LSD). Experiments were repeated independently for more than three times. All data are shown as the mean ± S.D. **P < 0.01, ***P < 0.001; ns, not significant
Fig 5: Overexpression of TNFAIP3 in ACSCs inhibits osteoclastogenesis in vitro. a, b TRAP staining (top) and immunofluorescence of TRAF6 (middle) and CTSK (bottom) in osteoclasts treated with 10% culture supernatant of transfected ACSCs. Knockdown of TNFAIP3 promotes OC formation, while overexpression of TNFAIP3 inhibits OC formation. Scale bars, 50 μm. c Quantification of TRAP+ multinucleated osteoclasts (> 3 nuclei) (left) and the relative fluorescence intensity of TRAF6 (middle) and CTSK (right) in (a–b). The statistical significance of differences was determined using one-way ANOVA with multiple comparison tests (LSD). Experiments were repeated independently for more than three times. All data are shown as the mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001
Supplier Page from Abcam for Recombinant human TNFAIP3 protein (Active) (DDDDK tag N-Terminus)