Figure 4

miR-101 function during IL-1β-induced chondrocyte extracellular matrix (ECM) degradation probably occurs through regulation of Sox9. Primary rat chondrocytes were transfected with miR-Scr, miR-101 mimic, and miR-101 inhibitor, and then treated with or without IL-1β 12 h post-miRNA transfection in the same manner as mentioned above. (A and B) Sox9 expression was analyzed by real-time PCR (A) and western blot (B) 24 h post miRNA mimic, inhibitor, or miR-Scr transfection. The right panel (B) is the densitometric analysis of Sox9 expression (left panel), normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The analysis was performed using images of three independent experiments, respectively; n = 3, *P < 0.05. (C and D) Primary rat chondrocytes were co-transfected with miR-Scr or miR-101 mimic together with Sox9 full-length vector (Sox9 expression vector containing both UTR and CDS regions) or Sox9 CDS vector (Sox9 expression vector containing only CDS region, no UTR). Sox9 expression was analyzed by real-time PCR (C) and western blot (D) 24 h post transfection; n = 3,*P < 0.05. (E and F) Primary rat chondrocytes were first transfected with miR-Scr or miR-101 inhibitor, and then siRNA against Sox9 (siSox9) or scrambled siRNA (siScr) were transfected. Sox9 expression was analyzed by western blot (E) 24 h post transfection. The lower panel (E) is the densitometric analysis of Sox9 expression (upper panel), normalized by GAPDH. The analysis was performed using images of three independent experiments, respectively; n = 3,*P < 0.05. (F) The sGAG content in the cell suspension was assessed by dimethylmethylene blue (DMMB) assay. Data were normalized by total protein content in the cell lysate in each group; n = 3,*P < 0.05.