2 deoxy d glucose 2 dg

A variety of different mouse lines including C57BL/6 wild‐type, EP4^−/−, Vav^CreEP4^fl/fl, and their appropriate control mice were anaesthetised by inhalation of isoflurane and the pro‐allergic cytokine IL‐33 (200 ng per treatment) was administered via intratracheal administration once daily for consecutive 3 days. When indicated, EP2 or EP4 agonist (each 10 μg) or vehicle control (PBS) were co‐administered with IL‐33. Indomethacin (5 mg/kg/day), PF‐04418948 (10 mg/kg/day) or vehicle (0.5% EtOH) was administered in drinking water started one day before IL‐33 administration. 2‐Deoxy‐d‐glucose (2‐DG, 1 g/kg/day) (Abcam) was administered via intraperitoneal injection (one injection per day throughout the whole experiment). All animals were culled via anaesthetic overdose by i.p injection of 200 μl Pentoject Pentobarbital Sodium solution (200 mg/ml) and subsequent exsanguination. Bronchoalveolar lavage (BAL) was collected (x3 lavages with 800 μl ice cold PBS) followed by aseptic lung dissection for subsequent tissue digestion. Both male and female mice were used in Figures 1E–G, 2 and 5N,O, while only female WT mice were used in Figures 1B,C, 3, 5.

Oligomycin (Abcam, ab141829, Cambridge, UK), 2-Deoxy-d-glucose (2-DG) (Abcam, ab142242, Cambridge, UK), glucose (Merck, Darmstadt, Germany), and Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (Abcam, ab120081, Cambridge, UK) were diluted in DMSO or ddH₂O according to manufacturer’s instructions. Cells were prepared according to the standard procedure described above. Drugs were added to the imaging medium and the plate incubated for 20 min on the device’s frame before starting the measurements. pO₂ measurements were performed in 20-s intervals for 30 min, and pH measurements in 10-s intervals for 30 min. After the measurements, nuclei were stained and microscopic images acquired as described above.

Rotenone, AICAR (5-aminoimidazole-4-carboxamide riboside), A-769662, metformin, ZLN024 and PT1 were purchased from Tocris and 2-deoxy-D-glucose (2DG) was purchased from Abcam. Oligomycin and antibodies to MLC, pMLC, pHSP27, pAMPKα, pACC were purchased from Cell Signaling Technology. Salicylate was purchased from Sigma. The anti-VE-cadherin and ROKα antibodies were from BD-Biosciences and the anti-RhoA antibody was from Merck Millipore. Glucose-free DMEM and dialysed serum were purchased from Gibco Life Technologies (Paisley, United Kingdom).