Effects of the mTOR inhibitor on iNOS expression elicited by TII. (A) Total cytosolic RNA was prepared from Control, or astrocytes treated with TII and rapamycin for different times, and used for Q-PCR analysis of iNOS expression. Data are expressed as fold change vs. each respective Control, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate, the experiment was repeated 2 times with similar results. Where indicated, 10 nM Rapamycin was added at the beginning of the experiment. In all time points TII significantly induced iNOS expression in comparison to Controls and rapamycin increases in 4 h the iNOS mRNA expression elicited by TII. Data are means ± SEM (n = 3). *P < 0.05, **P < 0.01, and *** P < 0.001 vs. TII; two-way ANOVA followed by Bonferroni's post-test. (B) Nitrite production elicited by TII was assessed at different time points, 10 nM rapamycin was added together with cytokines at beginning of experiment. Data are means ± SEM (n = 4). **P < 0.01, vs. Controls; and §§P < 0.01, vs. TII; two-way ANOVA followed by Bonferroni's post-test.