Figure 4

PUFAs strongly suppress pro-apoptotic action of EA during eATP stimulation. (a) RAW264.7 cells were pretreated with or without 200 µM EA and 50 µM DHA for 12 h, stimulated with 0.5 mM ATP for 6 h, and assayed for cell viability. Data shown are the mean ± SD (n = 3). ***p < 0.001. (b) RAW264.7 cells were treated with various concentrations of CFAs along with 200 mM EA for 12 h, stimulated with ATP at 0.5 mM for 6 h, and assayed for cell viability. Data are shown as relative cell viability (mean ± SD, n = 3), normalized with the viability of cells stimulated with ATP without any fatty acid. DHA: ***p < 0.001. EPA: ^##p < 0.01; ^###p < 0.001. AA: ^†††p < 0.001. LA: ^◇◇p < 0.01. OA: ^‡p < 0.05 (vs ATP+, EA+, CFA–). (c) BV2 cells were pretreated with or without 200 µM EA and 50 µM DHA for 12 h, stimulated with 0.5 mM ATP for 6 h, and assayed for cell viability. Data are shown as relative cell viability (mean ± SD, n = 3), normalized with the viability of cells stimulated with ATP without any fatty acid. **p < 0.01; ***p < 0.001; NS, not significant (vs ATP+, EA+, CFA–). (d) RAW264.7 cells were pretreated with or without 200 µM EA for 15 h, and treated with 50 µM DHA for the indicated time periods before stimulation with 0.5 mM ATP for 6 h. Cell viability was assessed, and data are shown as relative cell viability (mean ± SD, n = 3), normalized with the viability of cells stimulated with ATP without any fatty acid. ***p < 0.001; NS, not significant (vs ATP+, EA+, DHA–). (e) RAW264.7 cells were pretreated with 200 µM EA in the presence of either 5 µM DHA, GPR40 ligand (TAK-875, 10 µM) or GPR120 ligand (TUG-891, 50 µM), stimulated with 0.5 mM ATP for 6 h, and then assayed for cell viability. Data are shown as relative cell viability (mean ± SD, n = 3), normalized with the viability of cells stimulated with ATP without any fatty acid or ligand. ***p < 0.001; NS, not significant (vs ATP+, EA+, DHA or inhibitor–).