Hoechst h33258

Antibodies against phosphorylated p38, phosphorylated MKK3/6, MKK6, and MKK3b were from Cell Signaling Technology (Beverly, MA, USA). Antibodies against p38, HA, GFP, and Actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against RACK1 was from BD Biosciences (Franklin Lakes, NJ, USA). Glutathione-Sepharose beads, Protein A-Sepharose beads, Hanks’ balanced salt solution (HBSS), collagenase I, PI, hoechst (H33258), cycloheximide, and antibody against FLAG were from Sigma-Aldrich (St. Louis, MO, USA). p38 inhibitors SB203580 and SB239063 were from CalBiochem (San Diego, CA, USA). ECL chemiluminescence kit was from Amersham (Arlington Heights, IL, USA). In vitro protein translation system was from Promega (Madison, WI, USA).

Jurkat cells were treated with ISAE at the indicated concentrations for 24 h. Cells were then collected, washed with ice-cold PBS twice, and fixed in 4% paraformaldehyde for 20 min at 25 °C. Fixed cells were washed and resuspended in deionized water, and then a 10 µL cell suspension containing 5 × 10⁴ cells was spread on a microscope slide. Cell spreads were allowed to dry at room temperature for 30 min. The dried cell slides were used for the immuno-fluorescence assay. The slides were washed with washing buffer (0.1% BSA in PBS) and blocked with blocking buffer (5% BSA, 0.3% Triton X-100 in PBS) for 1 h at 25 °C. The slides were allowed to react with primary antibody (anti-p-H2A.X) at 4°C overnight, and then with tetramethyl rhodamine isothiocyanate-conjugated anti-mouse IgG antibody as secondary antibody for 1 h at room temperature. DNA was stained with Hoechst H33258 (Sigma-Aldrich) to localize the cell nuclei. Images of the nuclei and p-H2A.X were acquired and analyzed automatically using an HCS instrument (ImageXpress Micro System, Molecular Devices, Sunnyvale, CA, USA).

Dulbecco’s Modified Eagle’s Medium (DMEM), heat-inactivated fetal bovine serum (FBS), and H₂DCF-DA were purchased from Thermo scientific (Madison, WI, USA). Resazurin was purchased from Cayman Chemical (Ann Arbor, MI, USA). EPIXTRACT Nuclear Protein Isolation Kit (ENZ-45016) was purchased from Enzo Life Sciences (Hines Dr, Ann Arbor, MI, USA). Hoechst H33258 was purchased from Sigma-Aldrich (Burlington, MA, USA). α-Linolenic acid (ALA) and γ-linolenic acid (GLA) were purchased from ChemScene LLC (Monmouth Junction, NJ, USA). All other chemicals and reagents used were of analytical grade.