Plasminogen activator inhibitor type 1 (PAI-1) downregulated Toll-like receptor (TLR)2/6 expression and its signaling. (A) BV-2 microglial cells were treated with PAI-1 (100 ng/ml) for 5 hours. TLR2/TLR6 and dectin-1 gene expression was detected by reverse transcriptase PCR. β-actin was used as an internal control. (B) Alternatively, BV-2 microglial cells were treated with PAI-1 (100 ng/ml) for 24 hours. The levels of TLR2, TLR6, and TLR9 protein were then evaluated by western blotting analysis. α-tubulin was used as an internal control. Values indicate the results of densitometric analysis normalized to either β-actin or α-tubulin. (C) Primary microglia cultures were treated with mouse PAI-1 protein (100 ng/ml), lipopolysaccharide (LPS; 100 ng/ml), interferon (IFN)-γ; 50 U/ml), and lipoteichoic acid (LTA; 1 μg/ml) as indicated for 24 hours. (Upper panel) NO production was measured by a Griess reaction. (Lower panel) Cell viability was measured by 2,5-diphenyltetrazolium bromide (MTT) reduction assays, and the results expressed as the percentage of surviving cells over the control cells . Results are given as mean ± SD (n = 3). *P < 0.01, NS = not significant.