Fig. 10

The mTOR inhibitor rapamycin reduces LPS-induced-TNF-α production through preventing mTOR signaling-mediated mRNA translation. BV-2 cells were pretreated with vehicle or various concentrations of rapamycin for 30 min followed by treatment with 100 ng/ml LPS for 60 min (western blotting), 2 h (TNF-α mRNA), or 6 h (TNF-α protein). Western analysis (a) was performed as described in Fig. 8. Released TNF-α (b) was measured by ELISA. The expression of TNF-α mRNA (c) was quantified by real-time RT-PCR. Data are presented as mean ± SEM of three independent experiments. **p < 0.01 compared with LPS alone. The level of TNF-α in cells treated with LPS alone was 18.1 ± 2.3 ng/ml. d. LPS-induced NF-κB or TNF-α promoter activity is not altered by rapamycin. Following transfection of BV-2 cells with NF-κB or TNF-α promoter luciferase construct, cells were pretreated with various concentrations of rapamycin for 30 min prior to treatment with LPS (100 ng/ml) for 6 h. Luciferase activity is presented as a fold of control. Data are presented as mean ± SEM of three independent experiments