PD-L1 regulates directional persistent cell migration independently of PD-1. Related to Fig. 1. (A) Flow cytometry and quantification of cell surface PD-L1 in MCF-10A, PC3, and MDA-MB-231 cells. Data are shown as means ± SD from three independent experiments. Statistical significance was determined by two-sided unpaired t test. ****, 0.0001 > P. (B) Flow cytometry of cell surface PD-L1 in WT and PD-L1^KO PC3 cells. (C) Left: Flow cytometry of cell surface PD-L1 in PD-L1^KO and PD-L1^KO-PD-L1-EGFP MDA-MB-231. Right: PD-L1 expression was depleted in MDA-MB-231 and restored by expressing PD-L1-EGFP and protein levels were compared by immunoblotting. (D) Trans-well migration assays comparing cell migration in WT, PD-L1^KD, and PD-L1-EGFP expressing PC3 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. (E) Representative images of WT, PD-L1^KD, and PD-L1-EGFP PC3 cells during migration. Arrowheads track individual cell movements. Scale bars: 10 μm. (F) Threshold Cycle (CT) value from quantitative PCR results detecting the mRNA expression of GAPDH, PD-L1, and PD-1 in PC3 and MDA-MB-231 cells. Source data are available for this figure: SourceData FS1.