Fig 2: Live-cell multiphoton microscopy imaging of (a) FPR1-transfected HL-60 cells and (b) wild type HL-60 cells. The cells were incubated with probe 16 (1 μM) in HBSS buffer for 10 min on ice. Excess/unreacted probe 16 was removed from the media before imaging. Scale bar = 10 μm.

Fig 3: Binding poses of probes 4 (a), 7 (b), 16 (c), and 17 (d) with FPR1 (PDB ID: 7EUO).

Fig 4: Characterization of FPR1 endocytosis.A Schematic summarizing FPR1 trafficking and key regulatory steps before and after fMLF stimulation. B Schematic of the flow cytometry-based FPR1 internalization assay. Differentiated neutrophil-like HL-60 cells are stimulated with fMLF to induce receptor internalization at 37 °C. The remaining surface FPR1 is stained with an anti-FPR1 antibody on ice. The level of internalization is calculated relative to the FPR1 measurement of unstimulated cells. C Differentiated HL-60 cells were treated with 100 nM or 1 μM fMLF at different time-points (n = 2, shaded region indicates mean ± standard error). Dots represent different biological replicates in all figures. D Differentiated HL-60 cells were treated with different fMLF concentrations for 10 mins (n = 4). Bar height represents the mean values. E dHL-60 cells were either stimulated with AF488-fMLF or not stimulated for 5 min, fixed, and stained with an anti-FPR1 antibody (scale bar, 5μm).

Fig 5: Fomin activity is important for efficient FPR1 internalization.A Plot showing the effect of the knockout of major actin regulators on FPR1 surface levels in the basal (y axis) and post-stimulation surface expression screens (x axis). Effect of SMIFH2 on FPR1 internalization (B) in dHL-60s (20 μM, 50 μM, and 100 μM) and C in primary neutrophils (50 μM). Cells are untreated or pre-treated with SMIFH2 for 20 mins. (n = 6 for dHL-60s, n = 4 for primary neutrophils).