Figure 5

Janus kinase (JAK)/signal transducer and activator of transcription (STAT)-1 was involved in the plasminogen activator inhibitor type 1 (PAI-1)-enhanced microglial motility. (A) BV-2 microglial cells were treated with mouse PAI-1 protein (100 ng/ml) or interferon (IFN)-γ (50 U/ml), and cell lysates were collected at 30 minutes after the treatment. The levels of phosphorylated STAT1 (pSTAT1 at Tyr701) or total STAT1 protein were then evaluated by western blotting analysis. Ponceau S staining was performed to confirm the equal loading of the samples. (B) BV-2 microglial cells were transfected with control small interfering (si)RNA or low-density lipoprotein receptor-related protein (LRP)1 siRNA. The cells were harvested 48 hours after transfection and used for the experiments. Cells were treated with mouse PAI-1 protein (100 ng/ml) for 30 minutes. Phosphorylated STAT1 or total STAT1 was measured by western blotting analysis. α-tubulin detection was also performed to confirm the equal loading of the samples. Values indicate the results of densitometric quantification normalized to α-tubulin. (C) BV-2 microglial cells were transfected with control siRNA or LRP1 siRNA. The cells were harvested at 48 hours after transfection and then treated with mouse IFN-γ (50 U/ml) for 30 minutes. Phosphorylated STAT1 was detected by western blotting analysis. α-tubulin detection was also performed to confirm the equal loading of the samples. (D) BV-2 microglial cells were pretreated with AG490 (JAK-specific inhibitor; 20 μmol/l) for 30 minutes before the treatment with mouse PAI-1 protein (100 ng/ml), and then cell migration was evaluated by the wound-healing assay. Results are mean ± SD (n = 3). *P < 0.05, NS = not significant, compared with the untreated control (lower panel). Representative images are shown (upper panel; original magnification × 150).