Figure 1.
PD-L1 regulates directional persistent cell migration and invasion. (A) PD-L1 expression was depleted in MDA-MB-231 and 4T1 cells using CRISPR/Cas9, and protein levels were compared with WT cells by immunoblotting. The same α-tubulin immunoblot is shown in Fig. S3 B. (B) Flow cytometry of cell surface PD-L1 in WT and PD-L1KO MDA-MB-231 and 4T1 cells. (C) Trans-well migration assays comparing cell migration in WT and PD-L1KO MDA-MB-231 and 4T1 cells. Data are shown as means ± SD from three independent experiments. Statistical significance was determined by two-sided, unpaired t test. *, 0.05 > P ≥ 0.01; ****, 0.0001 > P. (D) Trans-well invasion assays comparing cell migration in WT and PD-L1KO MDA-MB-231 and 4T1 cells. Data are shown as means ± SD from three independent experiments. Statistical significance was determined by two-sided, unpaired t test. *, 0.05 > P ≥ 0.01; ***, 0.001 > P ≥ 0.0001. (E) Representative trajectories quantified based on the chemotaxis assay from three independent experiments: WT: n = 104 cells, PD-L1KO: n = 93 cells, and PD-L1KO-PD-L1-EGFP: n = 86 cells. (F) Rose plot diagrams show the direction of cell migration from the chemotaxis assay from three independent experiments: WT: n = 104 cells, PD-L1KO: n = 93 cells, and PD-L1KO-PD-L1-EGFP: n = 86 cells. (G) Quantification of the directionality of cells from the chemotaxis assay. Data are shown as means ± SD from three independent experiments. Statistical significance was determined by two-sided, unpaired t test. *, 0.05 > P ≥ 0.01; **, 0.01 > P ≥ 0.001. (H) Flow cytometry and quantification of cell surface PD-L1 in siCtrl and siPD-L1 MDA-MB-231. 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; ***, 0.001 > P ≥ 0.0001. (I) Trans-well migration assays comparing cell migration in siCtrl and siPD-L1 MDA-MB-231 cells. Data are shown as means ± SD from three independent experiments. Statistical significance was determined by two-sided, unpaired t test. *, 0.05 > P ≥ 0.01. Source data are available for this figure: SourceData F1.

PD-L1 regulates directional persistent cell migration and invasion. (A) PD-L1 expression was depleted in MDA-MB-231 and 4T1 cells using CRISPR/Cas9, and protein levels were compared with WT cells by immunoblotting. The same α-tubulin immunoblot is shown in Fig. S3 B. (B) Flow cytometry of cell surface PD-L1 in WT and PD-L1KO MDA-MB-231 and 4T1 cells. (C) Trans-well migration assays comparing cell migration in WT and PD-L1KO MDA-MB-231 and 4T1 cells. Data are shown as means ± SD from three independent experiments. Statistical significance was determined by two-sided, unpaired t test. *, 0.05 > P ≥ 0.01; ****, 0.0001 > P. (D) Trans-well invasion assays comparing cell migration in WT and PD-L1KO MDA-MB-231 and 4T1 cells. Data are shown as means ± SD from three independent experiments. Statistical significance was determined by two-sided, unpaired t test. *, 0.05 > P ≥ 0.01; ***, 0.001 > P ≥ 0.0001. (E) Representative trajectories quantified based on the chemotaxis assay from three independent experiments: WT: n = 104 cells, PD-L1KO: n = 93 cells, and PD-L1KO-PD-L1-EGFP: n = 86 cells. (F) Rose plot diagrams show the direction of cell migration from the chemotaxis assay from three independent experiments: WT: n = 104 cells, PD-L1KO: n = 93 cells, and PD-L1KO-PD-L1-EGFP: n = 86 cells. (G) Quantification of the directionality of cells from the chemotaxis assay. Data are shown as means ± SD from three independent experiments. Statistical significance was determined by two-sided, unpaired t test. *, 0.05 > P ≥ 0.01; **, 0.01 > P ≥ 0.001. (H) Flow cytometry and quantification of cell surface PD-L1 in siCtrl and siPD-L1 MDA-MB-231. 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; ***, 0.001 > P ≥ 0.0001. (I) Trans-well migration assays comparing cell migration in siCtrl and siPD-L1 MDA-MB-231 cells. Data are shown as means ± SD from three independent experiments. Statistical significance was determined by two-sided, unpaired t test. *, 0.05 > P ≥ 0.01. Source data are available for this figure: SourceData F1.

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