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. 2019 Mar 28;78(6):826–836. doi: 10.1136/annrheumdis-2018-214786

Figure 2.

Figure 2

CircSERPINE2 expression knockdown induced the expression of matrix-degrading enzymes in chondrocytes. (A) HCs were transfected with CircSERPINE2 siRNA or negative control siRNA at a final concentration of 20 nM. After 48 hours of transfection, the expression level of CircSERPINE2 was measured by RT-qPCR and normalised to β-actin level. n=9 (three different donors for three different experiments). *p<0.05. (B) HCs were transfected with CircSERPINE2#2 siRNA or negative control siRNA at a final concentration of 20 nM. After 48 hours of transfection, the expression level of SERPINE2 was measured by RT-qPCR and normalised to β-actin level. n=9 (three different donors for three different experiments). *p<0.05. (C) HCs were transfected with CircSERPINE2#2 siRNA or negative control siRNA at a final concentration of 20 nM. After 48 hours, cell apoptosis was detected with annexin V-FITC/PI double staining using quantitative FACS analysis. n=3 (three different donors). *p<0.05. (D) Cells were infected with CircSERPINE2 lentivirus or control virus. Overexpression was detected by RT-qPCR. n=9 (three different donors for three different experiments). *p<0.05. (E) HCs were infected with CircSERPINE2 lentivirus or control virus and stimulated with IL-1β for 48 hours. Cell apoptosis was detected with annexin V-FITC/PI double staining using quantitative FACS analysis. n=3 (three different donors). *p<0.05. (F–H) HCs were transfected with CircSERPINE2#2 siRNA or negative control siRNA at a final concentration of 20 nM, followed by the treatment with or without IL-1β. After 48 hours, WB for (F, n=3) MMP13, MMP3, aggrecan, SOX9, COL2A1 and ADAMTS4 detection; IF (G, scale bar: 50 µm) for MMP13, aggrecan, COL2A1 and ADAMTS4 expression detection; and RT-qPCR (H, n=9; three different donors for three different experiments; *p<0.05) for MMP3, MMP13, ADAMTS4, COL2A1, SOX9 and aggrecan expression evaluation were performed. FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; HCs, human chondrocytes; IF, immunofluorescence; IL; interleukin; PI, propidium iodide; RT-qPCR, real-time quantitative PCR; siRNA, small interfering RNA; WB, western blotting.