Skip to main content
. 2023 May 31;14:1188555. doi: 10.3389/fimmu.2023.1188555

Figure 3.

Figure 3

Influence of FPR ligands and inhibitors on proliferation, wound closure efficiency and migration speed. Relative proliferation of 24 h stimulated PHKs either treated with different concentrations of fMLF (A) or different concentrations of FPR2 ligands PSMα3 and MMK1 (B) compared to untreated (medium) control. Influence of FPR1 inhibitor tBOC (10 µM) on fMLF (50 nM) enhanced proliferation or FPR2 inhibitor WRW4 (1 µM) on PSMα3 (500 nM) or MMK1 (100 nM) induced proliferation (C). Representative phase contrast images of wound closure of undifferentiated N/TERT-1 keratinocytes treated with vehicle control (top row), 10 nM PSMα3 (middle row) or 10 nM PSMα3 plus 1 µM WRW4 (bottom row). Scale bar: 500 µm. Cyan line traces the wound edges (D). Percentage of gap (i.e., wound) area compared to the area of the whole field of view (y-axis) as a function of time (x-axis, h) (E). Mean migration speed (y-axis, μm/h) of N/TERT-1 cells as a function of time (x-axis, h) for cells repopulating the wound as in previous panels (F). Exemplary epifluorescence images of N/TERT-1 keratinocytes’ nuclei stained with live-cell DNA dye (Hoechst) during wound closure. Top row shows nuclei without tracks, while bottom row shows the same images with superimposed object detection (purple circles) and tracks (cumulative displacements of the nuclei). Green arrows mark a dividing cell (on top) and cell division as detected by the nuclear segmentation and tracking (bottom row, red arrows) (G). Using Trackmate Fiji plugin, cells were tracked for 12 h, split events were counted and divided by the total number of nuclei. Cells were treated with PSMα3 (10 nM), WRW4 (1 µM), both or vehicle control. Proliferation rate was normalized with respect to the mean of cells treated with vehicle control (H). Data represent mean and SEM of six independent experiments of three different donors, (A-C) or three independent experiments (D-H). *P < 0.05; **P < 0.01, significant difference versus the indicated FPR ligands as calculated by paired two-tailed Student’s t-test (C). A one-way ANOVA followed by a Dunnett’s multiple comparison test was performed and control (*P = 0.02) and PSMα3 plus WRW4 treated cells’ proliferation rate (*P = 0.02) was determined to be significantly altered compared to PSMα3 treated cells only (H).