Figure 7

Effect of NOV treatment on TNF-α-stimulated MMP-9 mRNA expression in DRG neuronal cultures. (A) DRG cultures were exposed to TNF-α (1 ng/ml) for 1 hour prior to NOV treatment (1 μg/ml). Cells were harvested 2, 5, 8 or 24 hours after NOV treatment. (B) DRG cultures were exposed to TNF-α (1 ng/ml) for 1 hour prior to NOV treatment (1 μg/ml) for 5 hours with recombinant human NOV protein produced using a baculovirus expression system in insect cells (bac) or with commercial human NOV protein produced in a murine cell line (mu). (C) Cells were infected with NOV adenovirus (Ad-NOV) or control virus for 48 hours and then exposed to TNF-α (1 ng/ml) for an additional 12 hours. MMP-9 mRNA expression was evaluated by RT-qPCR. Data reported are fold changes versus the corresponding control of DRG cultures exposed to normal medium (NT, not treated) and represent the mean of four values in a representative experiment out of two experiments giving similar results (*P < 0.05, **P < 0.01, ***P < 0.001 TNF-α + NOV versus TNF-α alone). (D) DRG cultures were exposed to TNF-α (1 ng/ml) for 1 hour prior to NOV treatment (1 μg/ml for 5 hours) 48 hours following transfection with a control non-silencing siRNA (Ctr) or a specific siRNA against integrin β₁, β₃ or β₅. Values represent the percentage of inhibition ± SEM of MMP-9 by NOV upon TNF-α stimulation compared to TNF-α exposure of two independent experiments (ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001 TNF-α + NOV versus TNF-α).