Fig. 4.

Coculture of MDM with A549 cells primes for activation of the p38 MAPK/MK-2 pathway that mediates increased 5-LO product synthesis. MDM were cocultured with A549 cells or kept in monocultures in a Boyden chamber (see Fig. 1a) for 48 h in the presence of IL-4. a MDM were separated and exposed to E. coli (O6:K2:H1; ratio = 1:50) for the indicated times. Cell lysates were prepared and immunoblotted for phospho-p38 MAPK, p38 MAPK, phospho-ERK-1/2, ERK-1/2, phospho-MK2, and normalized to β-actin or GAPDH for densitometric analysis. Data are means ± S.E.M., n = 3 separate donors. b MDM were preincubated for 15 min with 0.3 µM Skepinone-L or 0.1% DMSO (vehicle) and exposed to E. coli (O6:K2:H1; ratio = 1:50) for 90 min at 37 °C. LMs were extracted and analyzed by UPLC-MS–MS. Absolute amounts (pg/2 × 10⁶ MDM) of selected LM (PGE₂, LTB₄, 15-HETE and 12-HETE) are shown as bar charts. Data are means, n = 3. *P < 0.05, two-way ANOVA followed by Tukey post-hoc test