Fig. 6

GM1 dampens pro-inflammatory cytokines and NO production in Q140/140 and Q7/7 microglia. A Schematic experimental design and cytokine measurements. Q140/140 microglia were pre-treated with or without LPS (100 ng/ml) for 3 h, followed by washes and incubation with GM1 (50 µM) or vehicle (PBS) for 6 h. GM1 incubation significantly decreases the expression of Il-1b, Il-6 and Tnf (N ≥ 4). Gene expression was normalized over the housekeeping gene Ppia. TNF secreted in the conditioned medium (N = 5) was normalized over the total cell protein content of the cells in each well. Two-way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. B Microglia were treated with 100 ng/ml LPS for 3 h, followed by incubation with or without GM1 for 45 h. Levels of nitrite in the conditioned medium were normalized to total cell proteins. N ≥ 3. Two-way ANOVA with Sidak multiple comparison test. C Q140/140 microglia were polarized towards a pro-inflammatory phenotype by incubation with GM-CSF for 4 days, followed by 1 h priming with INF-γ and 48 h stimulation with LPS. GM1 or control vehicle were added for the last 24 h of incubation in LPS. Gene expression was normalized over Ppia. Ratio paired t-test. D Q7/7 and Q140/140 microglia were incubated with or without LTA (10 μg/ml) for 3 h, followed by washes and treatment with vehicle or GM1 (50 µM) for 8 h. GM1 significantly reduces the expression of Il-1b, Il-6 and Tnf mRNA. Gene expression was normalized over the geometric mean of three housekeeping genes (Normalization Index). Ratio paired t-test. Bars are mean values ± STDEV. *p < 0.05, **p < 0.01, ***p < 0.001