Medium for culturing the cell lines was purchased from Invitrogen. Other reagents were from Sigma. Dietary oils including olive, sesame, canola, fish oils were obtained from the Whole Foods brand 365. Unsaturated fatty acids (ALA, DHA, Erucic acid, EPA, oleic, LA, RA, and AA), Fluo-8 AM and lipase were obtained from Sigma (St. Louis, MO).
The mouse stromal cells expressing endogenous subtype EPs were isolated from mouse fat tissue and cultured using Dulbecco's Modified Eagle's Medium (DMEM) medium with fetal bovine serum (FBS) as described previously . The protocol for the animal fat cell extraction was approved by the University of Houston Institutional Animal Care and Use Committee.
EP1 receptor pcDNA construction
A pAcSG-EP cDNA cloned by our laboratory was first subcloned into EcoRI/XhoI sites of pcDNA3.1 (+) expression vector to generate the plasmid of pcDNA: human EP1. The pcDNA vector has a Cytomegalovirus (CMV) promoter and geneticin (G418) as the selection antibiotic.
Stable expression of recombinant human EP1 in HEK293 cells
The generation of the HEK293 cell line stably expressing human EP1 (HEK293-hEP1) has been described previously . Briefly, HEK293 cells cultured in DMEM containing FBS (10%), antibiotics, and antimycotics were transfected with the purified pcDNA: human EP1 using the Lipofectamine 2000 method . Approximately 48 hours after transfection, the cells were subcultured and incubated with G418 (selection antibiotic) for four weeks to generate the HEK293 cell line stably expressing human EP1 (HEK293-hEP1).
Western blot analysis
The cultured cells were collected and washed with PBS. The proteins were separated by 7-10% (w/v) SDS-PAGE under denaturing conditions and then transferred to a nitrocellulose membrane. Bands recognized by individual primary antibodies were visualized with horseradish peroxidase-conjugated secondary antibody as described .
Digestion of oils
One milliliter of each oil (olive, canola, sesame, and fish) was individually mixed with 10mg of lipase in 50 μl of PBS. The mixtures were vortexed and kept at 37°C for two hours and then centrifuged at 5,000 revolutions per minute (rpm) for five minutes. Supernatants were stored at 4°C.
Determination of PGE2 signaling
The cultured cells were washed twice (1 mL medium/well), and then incubated with new cell culture medium (1 mL/well) containing calcium binding reagent, Fluo-8 (50 μg). After 30 minutes of incubation at 37°C, the cells were washed with serum-free medium three times to remove the excess Fluo-8 that did not enter the cells, and then observed under a Nikon fluorescence microscope (Nikon, Eclipse Ti) using a software (NIS Elements 2.35) designed specifically for calcium signaling. To start the signaling assay, PGE2 (50 μL, 0.12 μM) was added. The increasing fluorescence signal, generated by the calcium bound to Fluo-8 inside the cells through the binding of PGE2 to its receptor, was recorded under the fluorescence microscope and analyzed.
Determination of the effects of the dietary oils on the PGE2-mediated receptor signaling in the cells
A procedure similar to that used above was performed for the determination of the effects of the dietary oils on PGE2-mediated receptor signaling. The only difference was that the PGE2 used was mixed with the digested individual oils at varied concentrations for 5 minutes before being added to the Fluo-8 loaded cells.
Determination of the effects of individual fatty acids on the PGE2-mediated receptor signaling in the cells
Individual fatty acids dissolved in DMSO (10 μl, 10 μM)) were mixed with the PGE2 for five minutes, and then added into Fluo-8-loaded cells. The fluorescence changes were recorded under the fluorescence microscope. The PGE2 mixed with PBS was used as control.
To observe the intensity of the fluorescence signaling mediated by the PGE2 in the absence and presence of the dietary oils and fatty acids, the specific effects of the dietary oils and fatty acids on the PGE2 signaling were identified by the comparison of the their signal intensities. After analyzing the control cells results, the results were plotted to show the difference between the oils and fatty acids. The experiments were repeated three times.
Student’s t-test was used as the statistical analysis tool.