Fig 1: LncRNA PART1 regulated cell growth, apoptosis and ECM-related genes through targeting miR-93-5p. (A) Images of NP cell colonies. The colony formation rates are shown on the right. (B) The apoptosis of NP cells was measured by flow cytometry. The apoptosis rates of NP cells are shown on the right. (C and D) The expression levels of Ki67 and C-caspase-3 in NP cells were measured by western blotting. (E-G) The expression levels of aggrecan, ADAMTS4, collagen II and MMP13 in NP cells were measured by (E and F) western blotting and (G) reverse transcription-quantitative PCR analysis. **P<0.001 vs. MC; ##P<0.001 vs. IC; and ^^P<0.001 vs. I. Blank, non-transfection; siNC, siRNA for negative control; siPART1, siRNA for PART1; MC, mimic for control; M, miR-93-5p mimics; IC, inhibitor for control; I, inhibitor for miR-93-5p; siPART1 + I, co-transfection with siRNA for PART1 and miR-93-5p inhibitor. ECM, extracellular matrix; NP, nucleus pulposus; MMP, matrix metallopeptidase.
Fig 2: Function of circCOG8 in disk NP cells under mechanical stress. (A,B) The human NP cells under compression treatment were transfected with the circCOG8 overexpression vector or circCOG8 shRNA, and the circCOG8 RNA level was examined using the RT-qPCR analysis. (C,D) The apoptosis ratio of the NP cells transfected with the circCOG8 overexpression vector or circCOG8 shRNA was determined by the flow cytometry analysis after the Annexin V-APC and 7-AAD double-staining. (E,F) The intracellular ROS levels of the NP cells transfected with the circCOG8 overexpression vector or circCOG8 shRNA were detected by DHE and measured by the flow cytometry analysis. (G,H) Human NP cells under compression were treated with the circCOG8 overexpression or circCOG8 shRNA, and the protein levels of Bax, Bcl-2, Aggrecan, Type II collagen, MMP-13, ADAMTS-4, and ADAMTS-5 were determined by the western blot analysis. The corresponding GAPDH band served as a common internal loading control for Bax, Bcl-2, Aggrecan, Type II collagen, MMP-13, ADAMTS-4, and ADAMTS-5. Data were represented as mean ± SD. *p < 0.05, **p < 0.01, n = 3 (Student’s t-test).
Fig 3: Manipulation of RORa via small molecular regulator and siRNA regulated chondrocyte metabolism.A Chemical structures of RORa antagonist SR3335 and agonist SR1078. B Primary chondrocytes were treated with different concentrations of SR3335 and SR1078 (0, 1, 5, 10 µM). After the indicated time points, viable cells were counted using CCK-8 assay. C Western blot analysis and quantification of COL2A1 and SOX9 expression, which are regulated by when treated with 1 µM SR3335 and SR1078 for 48 h. D The chondrocytes were treated with vehicle, 1 µM SR1078, or 1 µM SR3335 for 48 h. The mRNA level of COL2A1, SOX9, ADAMTS4, and MMP13 was determined with real-time PCR. E Western blot analysis of the indicated proteins in human chondrocytes treated with vehicle or the indicated concentration of compounds for 72 h. F The relative expression of RORA gene after infected with sh-RORA or sh-NC lentivirus for 72 h. G, H Western blot analysis and representative immunofluorescence images of indicated protein after sh-NC and sh-RORA infection. Scale bars, 20 µm. Representative blots and images (n = 3). The statistical data in B were analyzed with one-way ANOVA, data in D and F were analyzed with Student’s t test. *P < 0.05, **P < 0.01, ns = no significance. All data shown above are presented as mean ± SD.
Fig 4: mRNA expression levels of cartilage degradation factors and fibrogenic factors in rat condylar cartilage at 12 weeks following anterior cruciate ligament transection. Reverse transcription-quantitative polymerase chain reaction was used to detect the mRNA expression levels of cartilage degradation factors (A) MMP-3, (B) MMP-9, (C) MMP-13, (D) ADAMTS-4 and (E) ADAMTS-5, and the expression levels of fibrogenic factors (F) type III collagen, (G) TGF-ß1 and (H) ADAM-12. Data are presented as the mean ± standard deviation; *P<0.05 vs. Control; #P<0.05 vs. NS. ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; MMP, matrix metalloproteinase; NS, normal saline; TGF, transforming growth factor; VEGF, vascular endothelial growth factor.
Fig 5: Effect of CT on the expression of matrix degrading enzymes in IL-1ß-injured rat chondrocytes. (A) mRNA and (B) protein levels of MMP-13, MMP-3, MMP-9, ADAMTS4 and ADAMTS5 were assessed using reverse transcription-quantitative PCR and western blot analysis. **P<0.01 vs. Normal group; ##P<0.01 vs. IL-1ß group; &&P<0.01 vs. IL-1ß + CT (50 nM); $$P<0.01 vs. IL-1ß + IWR-1-endo. CT, calcitonin; IL-1ß, interleukin-1ß; MMP, matrix metallopeptidase; ADAMTS, ADAM metallopeptidase with thrombospondin type 1; Dkk-1, anti-dickkopf-1.
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