PD-L1 regulates front-rear polarity in migrating cells. (A) Fluorescent images of live WT or PD-L1^KO MDA-MB-231 cells show distribution of PH-AKT-GFP. Arrowhead indicates cell front. Scale bar: 10 μm. (B) Quantification of the percentage of cells with polarized distribution of PH-AKT-GFP in WT or PD-L1^KO MDA-MB-231 cells. Data are shown as means ± SD from three independent experiments. Statistical significance was determined by two-sided, unpaired t test. **, 0.01 > P ≥ 0.001. (C) Representative images show distribution of ezrin (green) and β4 integrin (red) in WT or PD-L1^KO MDA-MB-231 cells. Single channel only images are shown in black-white images. White arrowhead points to the cell rear. Scale bar: 10 μm. (D) Quantification of ezrin signal at the front and rear in the same cell in WT or PD-L1^KO MDA-MB-231 cells. Statistical significance was determined by two-sided, paired t test (n > 50 cells per condition, three independent experiments). **, 0.01 > P ≥ 0.001. (E) Representative images show distribution of p-ezrin (Thr567; green) and β4 integrin (red) in WT or PD-L1^KO MDA-MB-231 cells. Single-channel only images are shown in black-white images. White arrowhead points to the cell rear. Scale bar: 10 μm. (F) Quantification of p-ezrin (Thr567) signal at the front and rear in the same cell in WT or PD-L1^KO MDA-MB-231 cells. Statistical significance was determined by two-sided, paired t test (n > 50 cells per condition, three independent experiments). ****, 0.0001 > P.