Figure 3

Effects of mammalian target of rapamycin (mTOR) inhibitors on nitric oxide synthase (NOS)2 in microglia stimulated by both control-conditioned medium (C-CM) and CM from glioma cells activated with LPS/IFNγ (LI-CM). Cells were treated with C-CM alone or in combination with mTOR inhibitors. (A-B) and with LI-CM alone or in combination with mTOR inhibitors (C-D). Total cytosolic RNA was prepared from control, or microglial cells treated with the drugs for 4 h, and used for real time (Q)-PCR analysis of NOS2 expression. Data are expressed as fold change versus control, taken as calibrator for comparative quantitation analysis of mRNA levels. Each sample was measured in triplicate and the experiment was repeated three times with similar results. Data are means ± standard error of the mean (SEM), and were analyzed by one-way analysis of variance followed by Bonferroni post hoc test. ***P <0.001 versus control; °P <0.05 °°°P <0.001 versus C-CM and °P <0.05 °°P <0.01 versus LI-CM. (B-D) After 48-h incubation, the medium was used for nitrite assessment, whereas cells were lysed in 200 mM NaOH and protein content was evaluated by the Bradford method. Results are expressed as μM of nitrites/μg of proteins; data are means ± SEM (n = 6). *P <0.05, ***P <0.001 versus C-CM; *P <0.05 versus control and °P <0.001 °°°P <0.001 versus LI-CM.