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. Author manuscript; available in PMC: 2011 Mar 1.
Published in final edited form as: Osteoarthritis Cartilage. 2009 Nov 5;18(3):447–454. doi: 10.1016/j.joca.2009.10.007

Figure 5. Effect of chemical inhibitors of signaling pathways on HA oligosaccharide-mediated stimulation of pHAS2(1932)-Luc activity in transfected C-28/I2 cells.

Figure 5

Panel A. Diagrammatic representation of HAS-2 promoter construct, pHAS2(1932)-Luc. The transcriptional site is located near the end of the 5-untranslated region (5′ UTR). The translational start site (AUG) is located at the start of exon 2. The coordinates for the promoter represent bp position relative to the transcriptional start site. Panel B. C-28/I2 cells were first transiently transfected with pHAS2(1932)-Luc, reduced to serum-free conditions and then preincubated with or without the chemical inhibitors, PD98059, SB203580, Helenalin, Wortmannin (Wort) or LY294002, 30 minutes prior to, and during the addition of 250 μg/ml HA oligosaccharides (+) under serum-free conditions. The cells were rinsed, solubilized with passive lysis buffer and analyzed for luciferase activity using a luminometer. Values represent the average relative luciferase units (RLU) ± S.D. of data derived from triplicate cultures normalized to total protein. Fold increases following HA oligosaccharide treatment, in control (no inhibitor), PD98059, SB203580 and Helenalin conditions represented significant increases above control; p= 0.0248, 0.0002, 0.0013 and 0.0008, respectively as measured using an unpaired Student’s t test.