Figure 5. Effect of rapamycin on HMC-3 cell viability and protein synthesis. Microglial cells were stimulated with II for 24 h. RAPA in the concentration range of 0.1-10 nM was added at the beginning of the experiment. (A) At the end of experiments in which ROS production was assessed (Figure 4F), cells were lysed in 200 mM NaOH and protein content was evaluated by the Bradford's method. Results are pooled analysis of three independent experiments (n= 4-6 per experimental group). 1-10 nM RAPA significantly reduced HMC3 protein content, both in resting cells as well as in cells exposed to II. Data are means ± SEM (n=15) and were analyzed by one-way ANOVA followed by the Bonferroni's post hoc test. ***, P<0.001, versus Control. (B) The effect of different treatments on cell viability was assessed by the MTS reduction assay. II did not change HMC3 cell viability nor did treatment with RAPA. Results are shown as fold changes versus Control, set as 100 %. Results are pooled analysis of three independent experiments (n= 4 per experimental group). Data are means ± SEM (n=12) and were analyzed by one-way ANOVA followed by the Bonferroni's post hoc test. (C) The cytotoxic effect of the treatments was assessed by measuring the release of LDH in the incubation medium, taken as an index of cell toxicity. None of the treatments was toxic for HMC3 microglial cells. Results are shown as fold changes versus Control, whose optical density was set as 100 %. Results are pooled analysis of three independent experiments (n= 3-6 per experimental group). Data are means ± SEM (n=12) were analyzed by one-way ANOVA followed by the Bonferroni's post hoc test.