Effect of curcuminoids on cell-free microsomal prostaglandin E synthase-1 activity

Human mPGES-1 was pre-treated with DMSO or curcuminoids, the formation of PGE2 was initiated by the addition of PGH2 and the mPGES-1 product PGE2 was analyzed by RP-UV-HPLC.
Raw data including the absolute values (ng) and normalized data (% of control) of all individual experiments was uploaded.
The methods and results were published in Rao et al., Biochem Pharmacol. 2022 Sep:203:115202. doi: 10.1016/j.bcp.2022.115202 
mPGES-1 activity was measured in microsomal membranes of IL-1β-treated human lung adenocarcinoma epithelial A549 cells. A549 cells were incubated with IL-1β (2 ng/mL, Peprotech, Hamburg, Germany) in DMEM/high glucose (4.5 g/L) medium plus FCS (10 %) and penicillin/streptomycin (100 U/mL and 100 μg/mL; GE Healthcare, Freiburg, Germany) at 37 °C and 5 % CO2 for 48 h. Cell pellets were harvested and snap-frozen in liquid nitrogen. After resuspension and incubation in ice-cold homogenization buffer [potassium phosphate buffer (0.1 M, pH 7.4), phenylmethylsulphonyl fluoride (1 mM, Cayman Chemical), soybean trypsin inhibitor (60 μg/mL, Cayman Chemical), leupeptin (1 μg/mL, Cayman Chemical), glutathione (2.5 mM, Cayman Chemical) and sucrose (250 mM, Cayman Chemical)] for 15 min on ice, cells were sonicated (3 times, 20 s, each, at 4 °C). Differential centrifugation (first 10,000 × g, 10 min; then 174,000 × g, 60 min; at 4 °C) yielded the microsomal membrane fraction, which was resuspended in homogenization buffer and diluted in potassium phosphate buffer (0.1 M, pH 7.4) plus glutathione (2.5 mM, Cayman Chemical). 
To determine mPGES-1 activity, membranes (containing 2.5–5 μg total protein in 50 μL homogenization buffer) were pre-treated with vehicle (DMSO), test compounds or the mPGES-1 inhibitor MK-886 (10 μM, Cayman Chemical) for 15 min at 4 °C. The formation of PGE2 was initiated by the addition of PGH2 [20 μM in 50 µL potassium phosphate buffer (0.1 M, pH 7.4) containing phenylmethylsulphonyl fluoride (1 mM, Cayman Chemical), soybean trypsin inhibitor (60 μg/mL, Cayman Chemical), leupeptin (1 μg/mL, Cayman Chemical), glutathione (2.5 mM, Cayman Chemical)]. After 1 min incubation at 4 °C, the reaction was terminated with an equal volume of stop solution containing FeCl2 (40 mM) and citric acid (80 mM) as well as the internal standard 11β-PGE2 (1 nmol, Cayman Chemical). The mPGES-1 product PGE2 was extracted by solid phase extraction using Sep-Pak C18 35 cc Vac Cartridges (Waters), separated on Nova-Pak C18 Radial-Pak Column (4 μm, 5 × 100 mm, Waters) under isocratic conditions (30 % acetonitrile, 70 % water, 0.007 % trifluoroacetic acid) at a flow rate of 1 mL/min and detected at 195 nm. The mPGES-1 reference inhibitor MK-886 (10 μM, Cayman Chemical) inhibited PGE2 formation by 86.4 ± 0.5 %.