Reagents
ATP and H2O2 was purchased from Wako (Tokyo, Japan). LPS was purchased from Invivogen (San Diego, CA, USA). Aspirin was purchased from Tokyo Kasei (Tokyo, Japan). 1-Aminobenzotriazole, HET0016, Zileuton, PD146176, Celecoxib, MS-PPOH, ML355, and Triacsin C were purchased from Cayman (Ann Arbor, MI, USA).
Cell culture
RAW264.7 and BV2 cells, both obtained from ATCC, were cultured in RPMI 1640 and Dulbecco’s Modified Eagle Medium (Nakalai Tesque, Kyoto, Japan), respectively, containing 10% heat-inactivated fetal bovine serum (Sigma, Burlington, MA, USA) and 1% penicillin-streptomycin solution (Nakalai Tesque) in 5% CO2 at 37°C.
Preparation and treatment of fatty acids
OA (Nakalai Tesque), EA (Sigma), LA and LEA (Cayman), TVA (Olbracht Serdary Research Laboratories, Toronto, ON, Canada), and RA and PEA (Funakoshi, Tokyo, Japan) were prepared as described previously33. Briefly, fatty acids were dissolved in 0.1 N NaOH at 70 °C, and then conjugated with fatty acid-free BSA (Wako, pH 7.4) at 55 °C for 10 min to make 5 mM BSA-conjugated fatty acid stock solutions containing 10% BSA. Cells were treated with various concentrations of BSA-conjugated fatty acids by diluting stock solutions in a medium without fetal bovine serum (final BSA concentration was set to 1%).
Immunoblot analysis
Cells were lysed in ice-cold lysis buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton-X100, 10% Glycerol, and 1% protease and phosphatase inhibitor cocktail (Nacalai tesque). After centrifugation, the cell extracts were resolved by SDS-PAGE, and were analyzed as described previously35. The antibodies used for immunoblotting were against phospho-ASK1 (Thr-845), phospho-p38, p38 (Cell Signaling), β-actin (Santa cruz, Dallas, TX, USA), ASK1 (Wako), phospho-CaMKII (Thr-286), and CaMKII (gift from Dr. Kohji Fukunaga, Tohoku University)36. The blots were developed with ECL (Merck Millipore, Burlington, MA, USA), and detected with ChemiDoc Touch Imaging System (BioRad, Hercules, CA, USA).
Cell viability assay
Cell viability was assayed as described previously37. RAW264.7 and BV2 cells were seeded on 96-well plates. After any stimulation or treatment, cell viability was determined using Cell Titer 96 Cell Proliferation Assay (Promega, Madison, WI, USA), according to the manufacturer's protocol. The absorbance was read at 492 nm using a microplate reader (iMark microplate reader, Biorad). Data are normalized to control without stimulus, unless noted otherwise.
Bioimaging and quantification of intracellular ROS
Intracellular ROS was detected and quantified as described previously38. Briefly, after stimulation, cells seeded on a glass plate were treated with 10 μM DCFH-DA (Sigma) for 30 min, and intracellular ROS generation was observed using a Zeiss LSM800 laser confocal microscope (Carl Zeiss, Oberkochen, Germany) and the images were processed with Zen software. After background subtraction, ROS level was calculated by dividing total green fluorescence by cell numbers using Image J. Data are shown as mean ± SD of relative fluorescence intensity from three different fields of view.
Lipid analysis
Lipids were extracted by the method of Bligh and Dyer39. Isolated lipids were methylated with 2.5% H2SO4 in methanol. The resulting fatty acid methyl esters were then extracted with hexane and subjected to gas chromatography tandem mass spectrometry (GC-MS/MS) analysis. GC-MS/MS analysis was performed by using a GCMS-QP2010 Plus (Shimadzu, Kyoto, Japan) equipped with Zebron ZB-FAME 60m x 0.25mm x 0.20 µm (Phenomenex, Torrance, CA, USA). The oven temperature program was as follows: the initial temperature was 100 °C for 2 min, then raised to 280 °C at 15 °C/min, and held for 5 min. The injector and detector temperatures were set at 240 °C and 260 ºC, respectively. The amounts of incorporated TFAs were calculated based on standard curves created from serial dilution of the respective fatty acids, which was normalized with the amount of extracted protein measured by Bradford method using protein assay CBB solution (Nakalai Tesque).
Statistics
All the values are expressed as means ± SD, and statistical analyses were performed using GraphPad Prism software (v.9.3.0). All experiments were repeated at least three independent times. Two groups were compared using two-tailed Student’s t-test. Multiple-group comparisons were conducted using either the one-way ANOVA analysis or two-way ANOVA analysis followed by Tukey-Kramer or Dunnett’s test.