Fig. 5.

Time course of E. coli-induced lipid mediator release and lipoxygenase activation. Human M1 or M2 (5 × 10⁶ cells/ml PBS + Ca/Mg) was incubated with E. coli (O6:K2:H1; ratio = 1:50) at 37 °C for the times indicated. As control (ctrl), cells were incubated at 37 °C for 180 min (a) or 90 min (b, c) without E. coli. a Formed lipid mediators in M2 were isolated by SPE and analyzed by LC–MS–MS. Data are given as means ± S.E.M., n = 3 separate donors. b, c Subcellular redistribution of 5-LOX and FLAP in M1 and M2 (b) or 5-LOX and 15-LOX-1 in M2 (c). After exposure to E. coli for the indicated times, cells were fixed, permeabilized, and incubated with antibodies against 5-LOX (red), 15-LOX-1 (cyan-blue), and FLAP (green); scale bars = 5 µm. b In situ interaction of 5-LOX and FLAP in M1 and M2 (lower panel); scale bars = 5 µm (insets) and 10 µm (overview). Proximity ligation assay (PLA) was performed after exposure of cells to E. coli after the indicated times. DAPI (blue) was used to stain the nucleus and in situ PLA signals (magenta dots) visualize 5-LOX/FLAP interaction. Results shown for one single cell are representative for ~100 individual cells analyzed in n = 3 independent experiments (separate donors)