(A–E) Ca²⁺ imaging with the Fura-2AM indicator in HEK293 cells transfected with GPR37 cDNA or empty vector (mock transfection). (A) Representative images showing calcium responses (color changes) after TX14 (1 μM, 3 minute treatment) and NPD1 (30 nM, 3 minute [duration of treatment]) treatment. Scale bar: 50 μm. The pseudo-color scale (0–2) shows the possible range of calcium signaling. (B) Combined traces from 100 cells showing time-dependent iCa²⁺ increases induced by NPD1 and TX14 after GPR37 but not mock transfection. Arrows show the time points at which the images in A were collected. (C) Comparison of iCa²⁺ levels after 3 minutes of treatment with NPD1 (30 nM), TX14 (1 μM), RvD1 (100 nM), RvD2 (100 nM), RvE1 (100 nM), lipoxin (100 nM), DHA (1 μM), EPA (1 μM), and ionomycin (2 μM). *P < 0.05 versus baseline (vehicle); 2-way ANOVA followed by Bonferroni’s post hoc test. n = 3–4 cultures, with 73 to 340 cells analyzed for each condition. (D) Dose-response curves of NPD1- and TX14-induced iCa²⁺ increases. n = 3–4 cultures, with 72 to 250 cells analyzed for each condition. Note the different EC₅₀ values for these 2 compounds. (E) Inhibition of NPD1-induced (30 nM) iCa²⁺ increases by PTX (1 μg/ml, 16 h before treatment), thapsigargin (1 μM, 3 min), and EGTA (10 mM, 3 min). *P < 0.05; 1-way ANOVA. n = 3–4 cultures, with 131 to 186 cells analyzed per treatment. (F) Dot blots showing a dose-dependent binding of NPD1 and TX14, but not RvE1, to GPR37. The blots were coated with NPD1, TX14, and RvE1 and then incubated with cell lysates from HEK293 cells with GPR37 cDNA or mock transfection. (G–J) Ca²⁺ imaging with the Fluo-4AM Ca²⁺ indicator in WT and Gpr37^−/− pMΦ cultures. (G) Representative images showing Ca²⁺ responses (color changes) after NPD1 treatment (30 nM) in WT but not Gpr37^−/− mice. Pseudo-color scale (0–5) shows the possible range of calcium signaling. Scale bar: 50 μm. (H) Combined traces from 100 cells showing time-dependent iCa²⁺ responses after NPD1 treatment (100 nM, 3 min) in WT and Gpr37^–/– mice. (I) Comparison of iCa²⁺ levels after treatment with NPD1 (30 nM, 3 min), TX14 (1 μM, 3 min), PTX (1 μg/ml, 16 h), RvD1 (100 nM, 3 min), RvE1 (100 nM, 3 min), and ATP (100 μM, 3 min) in WT and Gpr37^–/– pMΦ cultures. *P < 0.05 versus Gpr37^–/–; ^#P < 0.05 (with PTX vs. without PTX); 2-way ANOVA followed by Bonferroni’s post hoc test. n = 3–4 cultures, with more than 300 cells analyzed for each condition. (J) Dose-response curves of NPD1- and TX14-induced iCa²⁺ increases in pMΦ cultures. n = 3 cultures, with 80–500 cells analyzed for each condition. Note the different EC₅₀ values for NPD1 and TX14. Data represent the mean ± SEM. F0,relative basal intensity; Fmax, relative peak intensity.