Fig. 2

AGR1.135 and AGR1.137 alter CXCL12-mediated CXCR4 dynamics and nanoclustering. Single-particle tracking analysis of JK−/− cells transiently transfected with CXCR4 (JK−/−-X4) treated with DMSO (control), AGR1.131, AGR1.135 or AGR1.137 on fibronectin (FN)- or FN +CXCL12-coated coverslips (DMSO: 581 particles in 59 cells on FN; 1365 in 63 cells on FN +CXCL12; AGR1.131: 1019 particles in 71 cells on FN; 1291 in 69 cells on FN +CXCL12; AGR1.135: 862 particles in 70 cells on FN; 1003 in 77 cells on FN +CXCL12; AGR1.137: 477 particles in 66 cells on FN; 566 in 64 cells on FN +CXCL12) n=3. (A) Frequency of CXCR4-AcGFP particles containing monomers plus dimers (≤2) or nanoclusters (≥3),± SEM, calculated from mean spot intensity values of each particle as compared with the value of monomeric CD86-AcGFP (n=3; n.s., not significant; ****p≤0.0001). (B) Intensity distribution (arbitrary units, a.u.) from individual CXCR4-AcGFP trajectories on unstimulated and CXCL12-stimulated JK−/−-X4 cells pretreated or not with the indicated compounds or vehicle (DMSO). Graph shows the distribution of all trajectories (gray outline), with the mean value of each experiment (black circles) and the mean of all experiments ± SD (red lines) (n=3; n.s., not significant; ***p≤0.001, ****p≤0.0001). (C) Percentage of mobile and immobile CXCR4-AcGFP particles at the membrane of cells treated as indicated. (D) Diffusion coefficients (D1–4) of mobile single particle trajectories at the membrane of cells treated as indicated, represented by box-and-whiskers plots (Tukey method) with median (black line) indicated. (n.s., not significant, ****p≤0.0001).