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. Author manuscript; available in PMC: 2018 Dec 15.
Published in final edited form as: Biochem Pharmacol. 2017 Sep 21;146:139–150. doi: 10.1016/j.bcp.2017.09.008

Figure 2. Effects of FFA4 agonism and antagonism on PMA-induced ROS generation in Raw 264.7 macrophages.

Figure 2

As described in Experimental Procedures, ROS generation was detected by suspending cells in HBSS containing the ROS-sensitive luminescent probe L-012 (100 μM) in the presence of the noted FFA4 modulators or their vehicles (veh). Basal luminescence was read for 5 min prior to addition of PMA (1 μM) or vehicle (H2O), after which, luminescence was recorded for an additional 90 min. In A-E, representative traces are shown for experiments repeated independently (n)-times. A, PMA induces rapid and robust ROS generation in Raw 264.7 cells. A representative trace is shown for this condition, which was included in all subsequent experiments (n = 24). B, The PKC inhibitor BIMII (10 μM, 20 min prior to PMA treatment) abolishes PMA-induced ROS generation (n = 3), demonstrating the PKC-dependency of NADPH-oxidase generation of ROS. C, The FFA4 agonist TUG-891 (3 μM, 1 h prior to PMA treatment) decreases PMA-induced ROS generation (n = 8). D, The FFA4 antagonist AH7614 (1 μM, 1 h prior to PMA treatment) increases PMA-induced ROS generation (n = 6). E, AH7614 blocks the ability of TUG-891 to reduce PMA-induced ROS generation, demonstrating that this effect is modulated directly by FFA4 receptor agonism (n > 5). F, Area under the curves (AUC) for all conditions induced by PMA were quantified (mean ± SEM) using Prism 3.0 and normalized to the maximal response elicited by PMA. G, Cell-free effects of TUG-891 and AH7614 on ROS shows no effects of either agent on ROS detection. Statistical analysis was performed by ANOVA and *** denotes p < 0.001 versus the maximal PMA-response, ††† denotes p < 0.001 versus the TUG-891-treated condition. H, The FFA4 agonist DHA (100 μM, 1 h prior to PMA treatment) decreases PMA-induced ROS generation (n = 3) and this effect was reversed by AH7614 (1 μM). The FFA4/FFA1 agonist GW9508, which has been shown to increase ROS generation via receptor-independent mechanisms robustly increases PMA-induced ROS generation.