Role of hypoxia-inducible factor-1 alpha in the regulation of plasminogen activator activity in rat knee joint chondrocytes G. Zhu, Y. Tang, X. Liang,

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Role of hypoxia-inducible factor-1 alpha in the regulation of plasminogen activator activity in rat knee joint chondrocytes G. Zhu, Y. Tang, X. Liang, M. Zheng, J. Yang, H. Zhou, L. Li, T. Qin Osteoarthritis and Cartilage Volume 17, Issue 11, Pages 1494-1502 (November 2009) DOI: 10.1016/j.joca.2009.05.005 Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 (A) The total RNA was extracted at 24h after transfection and the HIF-1α mRNA level was analyzed by quantitative real-time RT-PCR. The HIF-1α mRNA was normalized against β-actin and indicated % induction or reduction compared with HIF-1α mRNA levels in untreated cells under normoxia. Values are means±SD of three independent culture experiments. Statistically significant difference from normoxic untreated control values: *P<0.05. (B) The total protein was extracted at 48h after transfection, and the HIF-1α protein level was measured by Western blot. The β-actin levels were used as the internal controls. Values are means±SD of three independent culture experiments. (C) The growth curve of chondrocytes in different groups. HIF-1α-deficient chondrocytes under hypoxia had a significantly lower proliferation rate than the other cells. Values are means±SD of triplicate experiments in each group: *P<0.05. Osteoarthritis and Cartilage 2009 17, 1494-1502DOI: (10.1016/j.joca.2009.05.005) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 PA activity is given in [U/106 cells] and represents the sum of activities of uPA and tPA in cell lysates. Values are means±SD of triplicate experiments in each group. Statistically significant difference from normoxic untreated control values: *P<0.05. Osteoarthritis and Cartilage 2009 17, 1494-1502DOI: (10.1016/j.joca.2009.05.005) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 (A) The total RNA was extracted at 24h after transfection and the mRNA levels of uPA, tPA, uPAR and PAI-1 were analyzed by quantitative real-time RT-PCR. The mRNA levels were normalized against β-actin and indicated % induction or reduction compared with those in untreated cells under normoxia. Values are means±SD of triplicate experiments. Statistically significant difference from normoxic untreated control values: *P<0.05. (B) Intracellular/matrix-associate protein levels of uPA, tPA, uPAR, and PAI-1 were measured by Western blot, and normalized by β-actin at 48h after transfection. (C) The soluble protein levels of uPA, tPA and PAI-1 in the supernatant were analyzed by ELISA analysis. The soluble protein levels were determined in triplicate wells and normalized to total protein. Statistically significant difference from normoxic untreated control values: *P<0.05. Osteoarthritis and Cartilage 2009 17, 1494-1502DOI: (10.1016/j.joca.2009.05.005) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 To detect binding of HIF-1 to the PAI-1 promoter, a CHIP assay was performed with the PAI-1 promoter fragment containing the HRE and a promoter fragment containing no HRE as a control (non-HRE). Input DNA that is not enriched by immunoprecipitation is amplified as positive control. Osteoarthritis and Cartilage 2009 17, 1494-1502DOI: (10.1016/j.joca.2009.05.005) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 To evaluate the role of HIF-1α in IL-1β-mediated regulation of PAI-1, cells were treated by IL-1β alone or combined with siRNA2 under normoxia and hypoxia respectively. (A) The total RNA was extracted at 24h after treatment, and the levels of HIF-1α and PAI-1 were analyzed by real-time RT-PCR and normalized against β-actin. Statistically significant difference from untreated control values: *P<0.05. (B) The total protein was extracted at 48h after treatment, and HIF-1α and PAI-1 protein levels were measured by Western blot. The β-actin levels were used as the internal controls. Values are means±SD of three independent culture experiments. Osteoarthritis and Cartilage 2009 17, 1494-1502DOI: (10.1016/j.joca.2009.05.005) Copyright © 2009 Osteoarthritis Research Society International Terms and Conditions