Fig. 5 |. AKBA modulates LM formation in 5-LOX-expressing HEK293 cells.

Stably transfected HEK293 cells (1 × 10⁶) were pretreated with 25μM AKBA (blue), 1 μM NDGA (white) or vehicle (0.1% DMSO, black) for 5 min before stimulation with 2.5 μM A23187 plus 1 μM AA for another 15 min at 37°C. Cell supernatant was subjected to solid-phase extraction and UPLC-MS/MS analysis, and LM are shown in pg per 1 × 10⁶ cells of n = 3 independent experiments. a, Effect of AKBA and NDGA on the LM profile of 5-LOX and FLAP-expressing cells. Data were log-transformed for statistical analysis *P < 0.05, **P < 0.01 AKBA versus vehicle control; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 NDGA versus vehicle control, ANOVA and Dunnett’s multiple comparisons test. b, Effect of AKBA and NDGA on 12-HETE biosynthesis by other LOX isoforms expressing cells. c,d, 5-LOX translocation in HEK293 expressing 5-LOX (c) or 5-LOX and FLAP (d). Cells were preincubated for 5 min with 25 μM AKBA, 1 μM NDGA or vehicle (0.1% DMSO) and then stimulated with 2.5 μM A23187 for another 15 min. Then, cells were fixed, permeabilized and incubated with antibodies against 5-LOX (red) and FLAP (green). Scale bars, 10μm. Results shown for one single cell are representative for approximately 100 individual cells analyzed in n = 3 independent experiments.