FAM3D is an agonist in FPR1- or FPR2-mediated chemotaxis. (A,B) The chemoattractant activity of HEK293 cells expressing FPR1 or FPR2 cells towards FAM3D. In a Boyden chamber system, the chemoattractant effects of various concentrations of recombinant FAM3D (1, 10, 100 nM) or fMLF, WKYMVm were tested for their ability to chemoattract HEK293 cells expressing FPR1 or FPR2, and the chemotaxis index and the significant difference compared with chemotaxis towards medium were calculated. The cells were pretreated with or without 5 μM cyclosporine H or 10 μM WRW4 for 1 h. (C,D) FAM3D (2, 20, 200, or 2000 ng/ml) or CXCL8 (200 ng/ml) was tested for its ability to chemoattract HEK293 cells expressing CXCR1 or CXCR2 cells, and the chemotaxis index and the significant difference compared with chemotaxis towards medium were calculated. (E,F) Mouse monoclonal antibodies targeting FAM3D (5E12 and 6D7) were applied in the chemotaxis assay. FAM3D (20, 200 or 2000 ng/ml) was pretreated with 5E12 or 6D7 (10 or 50 μg/ml) for 30 min, and the chemotaxis index was calculated for HEK293 cells expressing FPR1 or FPR2. (G,H) FAM3D (10 nM) was tested for its ability to chemoattract HEK293 cells expressing FPR1 or FPR2 with or without PTX pretreatment (1, 10 or 100 ng/ml) at 37°C for 30 min, and the chemotaxis index was calculated. Results are mean±s.e.m. (n=3). *P<0.05, **P<0.01 and ***P<0.001 (Student's t-test).

FAM3D stimulates internalization of FPR1. (A) BSA, FAM3D or fMLF (100 nM or 1000 nM) was used to stimulate HEK293 cells expressing FPR1–EGFP for 1 h. Cells were then fixed and analyzed by confocal microscopy. Scale bars: 25 μm. (B) Flow-cytometric analysis using anti-FPR1 as the primary antibody to detect the quantity of FPR1 on the cell surface after the stimulation with BSA, FAM3D or fMLF (100 nM or 1000 nM) for 1 h. (C) HEK293 cells expressing FPR1 were stimulated by 100 nM or 1000 nM fMLF, FAM3D or BSA, and the quantity of FPR1 on the cell surface was evaluated by flow cytometry and the rate of FPR1 internalization (mean±s.e.m.) was calculated for three independent experiments. *P<0.05 and **P<0.01 (Student's t-test).

FAM3D stimulates internalization of FPR2. (A) BSA, FAM3D or WKYMVm (10 nM or 100 nM) was used to stimulate HEK293 cells expressing FPR2–EGFP for 1 h. Cells were then fixed and analyzed by confocal microscopy. Scale bars: 25 μm. (B) Flow-cytometric analysis using anti-FPR2 as the primary antibody to detect the quantity of FPR2 on the cell surface after the stimulation with BSA, FAM3D or WKYMVm (10 nM or 100 nM) for 1 h. (C) HEK293 cells expressing FPR2 were stimulated by stimulated by 10 nM or 100 nM WKYMVm, FAM3D or BSA, and the quantity of FPR2 on the cell surface was evaluated by flow cytometry and the rate of FPR2 internalization (mean±s.e.m.) was calculated for three independent experiments. *P<0.05, **P<0.01, and ***P<0.001 (Student's t-test).

FAM3D can induce FPR1- or FPR2-mediated Ca²⁺ flux in HEK293 cells transiently transfected with FPR1 or FPR2. (A,B) HEK293 cells expressing FPR1 were loaded with 10 μM fluo-3 AM for 0.5 h. After cells were washed with PBS three times and images of the unstimulated state were obtained by confocal microscopy, cells were stimulated by FAM3D (200 nM) or fMLF (10 nM) (first arrow in graph) and constantly observed for 1.5 min. The second stimulation was then added (second arrow in graph), and the cells were constantly observed for another 1.5 min. The fluorescence intensity of the cells before and after the first and second stimulation was evaluated and analyzed by the Leica System Analysis Software. (C,D) HEK293 cells expressing FPR2 were loaded with 10 μM fluo-3 AM for 0.5 h. After cells were washed with PBS three times and images of the unstimulated state were obtained by confocal microscopy, cells were stimulated by FAM3D (200 nM) or WKYMVm (10 nM) and constantly observed for 1.5 min. The second stimulation was then added, and the cells were constantly observed for another 1.5 min. The fluorescence intensity of the cells before and after the first and second stimulation was evaluated and analyzed by the Leica System Analysis Software. Scale bars: 25 μm. Results are means (n values are shown on the figures).

RBAs confirm the interaction of FPR1 or FPR2 with FAM3D. (A) RBA for HEK293 cells expressing FPR1. In the saturation experiments, equivalent quantities of ¹²⁵I-FAM3D were incubated with varying quantities of FPR1 cell membrane extract in binding buffer. The y-axis represents the radioactivity of the binding, and the x-axis represents the logarithmic form of the concentration of FAM3D. In the competitive binding assays, equivalent quantities of cell membrane extract and ¹²⁵I-FAM3D were incubated with varying quantities of unlabeled FAM3D, fMLF, cyclosporine H or CCL2. The y-axis represents the radioactivity of the specific binding complexes, and the x-axis represents the logarithmic form of the concentration of the competitors. (B) RBA for HEK293 cells expressing FPR2. In the saturation experiments, equivalent quantities of ¹²⁵I-FAM3D were incubated with varying quantities of FPR2 cell membrane extract in binding buffer. The y-axis represents the radioactivity of the binding, and the x-axis represents the logarithmic form of the concentration of FAM3D. In the competitive binding assays, equivalent quantities of cell membrane extract and ¹²⁵I-FAM3D were incubated with varying quantities of unlabeled FAM3D, WKYMVm, WRW4 or RS102895. The y-axis represents the radioactivity of the specific binding complexes, and the x-axis represents the logarithmic form of the concentration of the competitors.

Flow-cytometric analysis of the cellular composition of peritoneal cellular recruitment, and experiments showing FAM3D activates ERK1/2 and p38 signaling in mouse neutrophils. (A,B) Flow-cytometric analysis of the peritoneal cellular recruitment 6 h after intraperitoneal injection of PBS or FAM3D. The x-axis represents CD11b, and the y-axis represents Ly6G. (C,D) Flow-cytometric analysis of G1 or G2 from Fig. 7B. The x-axis represents FPR1, and the y-axis represents FPR2. (E) Cells were stained for flow cytometry with fluorochrome-conjugated monoclonal antibodies specific for macrophages (F4/80), dendritic cells (CD11c), T lymphocytes (CD3) and B cells (B220) and the number of each category was calculated. Data are mean±s.e.m. (n=3). *P<0.05 (Student's t-test). (F) Activation of ERK1/2 (pERK, phosphorylated ERK1/2) and p38 signaling (p-P38, phosphorylated p38 MAPK proteins) as assessed by western blotting in freshly isolated neutrophils treated with 200 ng/ml FAM3D over a 2- or 5-min timecourse and pretreated with or without 5 μM cyclosporine H or WRW4 for 1 h.