Fig. 3.
Signaling pathways mediating bacteria-induced 5-LOX translocation in HEK293 cells. Subcellular localization of 5-LOX was determined by immunofluorescence microscopy. a HEK293 cells stably expressing 5-LOX (stained in red) and FLAP (green) were pre-incubated with 0.1% DMSO (vehicle), with the p38-MAPK inhibitors skepinone-L (0.3 µM) or SB203580 (1 µM) for 10 min at 37 °C and stimulated with 10% BCM derived from S. aureus (LS1) for the indicated periods. b HEK293 cells stably co-expressing FLAP with the 5-LOX_S271A mutant were stimulated as described in a. Microscopic images are given as overlay of 5-LOX and FLAP staining. c Stable expression of 5-LOX, 5-LOX_S271A, and FLAP in HEK293 cells was verified by immunoblotting. Images are representative for three independently collected cell lysates. Intracellular Ca2+ concentration of Fura-2AM-labeled HEK293 cells was monitored upon stimulation with d 10 µg/mL α-hemolysin or with e 10% of unconditioned medium (vehicle) or BCM of S. aureus strains (LS1, LS1∆hla or LS1∆agr/sarA) under continuous fluorescence reading. 2 µM ionomycin was used as positive control. Results are given as mean ± SEM. #p < 0.05, ##p < 0.01, and ###p < 0.001 LS1 or ionomycin vs. vehicle control or *p < 0.05, **p < 0.01 and ***p < 0.001 vs. the corresponding LS1 wild-type strain using one-way ANOVA with Tukey multiple comparison post hoc test. f 5-LOX translocation was detected by immunofluorescence microscopy in HEK293 cells stably expressing 5-LOX (red) and FLAP (green) after 10 min of pre-incubation with or without 10 mM EDTA in PBS+/+ followed by incubation for 90 min with 10 µg/mL α-hemolysin or 10% BCM of S. aureus (LS1). Microscopic images are given as overlay of 5-LOX and FLAP staining. Results shown are representative for at least three independent experiments