Fig. 6 | Nature Communications

Fig. 6

From: Human macrophages differentially produce specific resolvin or leukotriene signals that depend on bacterial pathogenicity

Fig. 6

E. coli elevates [Ca2+]i in M1 and M2 and Ca2+ is required for LOX translocation. a Measurement of [Ca2+]i. Fura-2/AM-loaded M1 or M2 in PBS containing 1 mM Ca2+ was treated with 1 µM fMLF, 2 µM ionomycin or vehicle (0.1% DMSO) for 10 min or with E. coli (O6:K2:H1), attenuated E. coli (O6:K2:H1), or non-pathogenic E. coli strain BL21 (ratio 1:50, each), or PBS + Ca/Mg as vehicle at 37 °C for up to 90 min. The ratio of absorbance at 340 vs. 380 nm reflecting [Ca2+]i is given as percentage of cells that were lysed with Triton X-100 (=100% control). Data are given as means ± S.E.M., n = 4 separate donors. *P < 0.05; **P < 0.01; ***P < 0.001 vs. t = 0 s or min, one-way ANOVA with Tukeyʼs multiple comparison test. b Subcellular redistribution of 5-LOX and FLAP in M1 and M2 (upper panel) or 5-LOX and 15-LOX-1 in M2 (lower panel) in the presence or absence of Ca2+. M1 or M2 (1 × 106 cells/ml) was incubated with E. coli (O6:K2:H1) or non-pathogenic E. coli strain BL21 (ratio 1:50, each) in PBS + Ca/Mg or PBS plus 0.5 mM EDTA and 20 µM BAPTA/AM at 37 °C as indicated. Cells were then fixed after 90 min, permeabilized, and incubated with antibodies against 5-LOX (red), 15-LOX-1 (cyan-blue), and FLAP (green); scale bars = 5 µm. Results shown for one single cell are representative for ~100 individual cells analyzed in n = 3 independent experiments (separate donors)

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