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. 2020 Sep 18;11:550824. doi: 10.3389/fimmu.2020.550824

FIGURE 4.

FIGURE 4

The CXCL12/HMGB1 heterocomplex-mediated chemotaxis requires both β-arrestin1 and β-arrestin2. (A) Cell migration assay. Wt and β-arrestin KO HeLa cells were allowed to migrate in response to the indicated concentrations of CXCL12 and/or HMGB1 for 18 h. One representative spider plot, out of three independent experiment performed, is shown for each experimental condition. Trajectories of at least 50 single tracked cells migrating following (black) or moving in the opposite direction (red) of the gradient, are shown. Black and red dots in the plots represent the final position of each single tracked cell. (B,C) Quantitative evaluation of the Forward Migration Index (FMI) after stimulation with the CXCL12/HMGB1 heterocomplex (B) or with 100 nM CXCL12 (C) in wt and β-arrestin KO HeLa cells. Mean ± SEM of at least 3 independent experiments, tracking n = 25÷50 cells. (D–F) Quantitative evaluation of the accumulated distance after stimulation with the vehicle only (D), the CXCL12/HMGB1 heterocomplex (E), or with 100 nM CXCL12 (F) in wt and β-arrestin KO HeLa cells. Data are shown as pooled mean ± SEM of n = 181; 150; 81; 136 (D); n = 178; 230; 108; 150 (E), n = 179; 230; 118; 150 (F) cells analyzed. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, using one-way ANOVA followed by Tukey’s multiple comparison test.