Fig. 5

Stimulation of the ganglioside biosynthetic pathway with L–t-PDMP decreases pro-inflammatory microglia activation. A BV2 cells were incubated for 72 h with the indicated concentrations of L–t-PDMP to increase ganglioside synthesis. A representative dot-blot and quantification of cellular ganglioside levels before and after treatment are shown (N = 3). B Representative histogram and relative flow cytometry quantification (mean fluorescence intensity, MFI) of TLR-4 present at the plasma membrane of BV-2 cells after treatment with 10 µM L–t-PDMP for 72 h. C Representative immunoblot showing phospho-IKK and phospho-p38 MAPK levels in BV2 cells stimulated with LPS (100 ng/ml) for the indicated time, after cell treatment with L–t-PDMP (15 µM, 72 h). The numbers under the blots show fold-change over untreated control, after normalization for total IKK or p38-MAPK levels. The experiment was repeated twice with similar results. D Expression of TNF and IL-1β mRNA in BV-2 cells treated as indicated above (N = 3). E Dot-blot analysis of ganglioside levels in murine primary microglia incubated with L–t-PDMP (10 µM for 72 h). F Expression of IL-1β and TNF mRNA in primary microglia treated for 72 h with 10 µM L–t-PDMP and stimulated with the indicated concentrations of LPS for 6 h (N = 3). G TNF secretion by murine microglia after treatment with L–t-PDMP and stimulation with the indicated concentrations of LPS (N = 3). Data shown are mean values ± STDEV. One-way ANOVA with Sidak’s multiple comparisons test was used in A; two-tailed t test was used in B and E; two-way ANOVA with Tukey’s multiple comparisons test was used in D, F and G. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001