Fig. 6
Effects of DHA and/or LPS treatment on 4-HHE and 4-HNE levels in microglia cells. a, b Cells were cultured in 60-mm dish and serum-starved for 3 h before addition of DHA (10, 25, and 50 μM) for 1 h and LPS (100 ng/mL) for 6 h. After treatment, culture medium was removed and cells were suspended with 0.5 mL of PBS:H2O (1:1, v/v). Cell suspension was vortexed and aliquots taken for LC-MS/MS measurement as well as protein assay as described in the “Methods” section. Results depict levels of 4-HHE (a) and 4-HNE (b) upon treatment with DHA and expressed as picogram per 10 μg of protein. Each value represents the mean ± SEM of three biological replicates with duplicate analysis. Analyzed by one-way ANOVA followed by Bonferroni post-tests; “a” represents significant differences (p < 0.05) comparing DHA groups with control group. Levels of 4-HHE (c) and 4-HNE (d) upon treatment of cells with LPS (100 ng/mL) and/or DHA (50 μM). One-way ANOVA followed by Bonferroni post-tests; “a” significant differences (p < 0.05) comparing test group with control group, and “b” represents significant differences (p < 0.05) comparing LPS group with DHA and DHA + LPS groups. e Protein concentration from four passages of microglia cells indicated no significant changes due to the different treatment conditions