FIGURE 6.

TNF‐α suppresses chondrogenesis of BMMSCs by downregulating YAP, while aspirin reverses these TNF‐α‐induced effects through stabilization of YAP. (A) The mRNA levels of YAP and key Hippo pathway target genes, including CTGF and CYR61, were measured by qPCR. (B) The p‐YAP and YAP protein expression levels were examined using western blot assays. (C, D) Stable overexpression of YAP‐FLAG or vector in BMMSCs was conducted. qPCR (C) and western blot (D) assays were performed to ensure overexpression efficiency. Chondrogenesis was then induced in BMMSCs for 14 days with or without TNFα, and expressions of YAP, COL2A1, ACAN, SOX9, ADAMTS4, MMP9, and MMP13 were detected by qPCR (E) and western blot (F). (G, H) Expressions of CTGF, CYR61 and YAP in different groups were examined by qPCR (G). The p‐YAP and YAP protein expression levels were assessed by western blot (H). (I, J) YAP expression was silenced in BMMSCs by transduction with lentivirus encoding YAP‐shRNA. Scramble‐shRNA was used as the control. qPCR (I) and western blot (J) were conducted to determine the silencing efficiency. Then, chondrogenesis differentiation was induced in BMMSCs for 14 days with or without vehicle, 10 ng/ml TNFα, and 100 μmol/L aspirin, and the expressions of YAP, COL2A1, ACAN, SOX9, ADAMTS4, MMP9, and MMP13 were detected using qPCR (K) and western blot (L) analyses. Values in A, C, E, G, I, and K are the mean ± SD of three independent experiments. ACAN, aggrecan; ADAMTS4, ADAM metallopeptidase with thrombospondin type 1 motif 4; COL2A1, collagen type II alpha 1 chain; CTGF, connective tissue growth factor; CYR61, cysteine‐rich angiogenic inducer 61; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; MMP9, matrix metallopeptidase 9; MMP13, matrix metallopeptidase 13; SOX9, SRY‐box transcription factor 9; YAP, yes‐associated protein. *p < 0.05, **p < 0.01, ***p < 0.001