Increased nitric oxide (NO) production fostered by Cp treatment in combination with lipopolysaccharide (LPS) depends on an incremented inducible nitric oxide synthase (iNOS) activity. (A) Nitrite production assessed by Griess assay in culture medium of microglial cells after 24 hours of treatment with medium (Ctrl), LPS (10 ng/ml) alone and in combination with Cp (20 μg/ml) or the same treatments performed following 1 hour pre-treatment with increasing concentrations of L-NAME (0.1, 0.25 and 1 mM). Results are expressed as μM of nitrite present in culture medium that reflects the NO production. Reduction percentage of nitrite production induced by L-NAME pre-treatment is indicated. (B) Western blot analysis of iNOS expression in microglial cells after treatments as in (A). Densitometric optical density (OD) for iNOS bands was normalized with α-tubulin expression and was reported as ratio of the OD of specific treatments versus OD of LPS treatment. Bottom panels are representative of one experiment. (C) Measure of iNOS activity in the lysate of microglial cells after LPS and LPS + Cp treatment. Results are reported as the ratio of nitrite and nitrate production (μM) in the lysate of cells treated with LPS + Cp versus LPS alone. Nitrite production was normalized by iNOS and α-tubulin expression levels as revealed by Western blot densitometric analysis. (D) Representative Western blot analysis of iNOS and α-tubulin expression in lysates of microglial cells treated with LPS and LPS + Cp that were used for enzyme activity normalization. Three/four independent experiments were performed (as indicated n =) and mean values, calculated using pooled data from different experiments, with standard error are reported. Statistical P-values were evaluated by non-parametric Mann-Whitney test. In all analyses, P < 0.05 was considered to be statistically significant.