Figure 2.
PD-L1 concentrates at the cell rear and promotes rear retraction generating PD-L1 containing retraction fibers and migrasomes. (A) Left: Representative time-lapse microscopy images of PD-L1KO-PD-L1-EGFP MDA-MB-231 cells show PD-L1 expression during the formation of retraction fibers and migrasomes. Scale bar: 10 μm. Right: Quantification of PD-L1 signal from the rear toward inner cell body over time. Arrowheads indicate retracting cell rear. (B) Representative time-lapse microscopy images of 4T1-PD-L1-EGFP cells show PD-L1 localizes at retraction fibers and migrasomes. Scale bar: 10 μm. (C) Representative time-lapse microscopy images of PD-L1-EGFP PC3 cells show PD-L1 localizes at retraction fibers and migrasomes. Scale bar: 10 μm. (D) A representative image of WT MDA-MB-231 cells stained with F-actin (phalloidin, green) and PD-L1 (red). Co-localization pixels of PD-L1 and F-actin are shown in the black-white image on the bottom right. Scale bar: 10 μm. (E) Images of PD-L1-EGFP (green) and WGA (red) in migrating cells. The boxed region outlines retraction fibers and migrasomes. Single-channel images are shown as black-white images. Scale bar: 10 μm. (F) Representative time-lapse microscopy images of WT (top) or PD-L1KO (bottom) MDA-MB-231 cells pre-labeled with SiR-Actin. Scale bar: 10 μm. Arrowheads point to cell rear and retraction fibers. (G) Quantification of retraction fiber length from images of WT or PD-L1KO MDA-MB-231 and 4T1 cells. Data are shown as means ± SD from three independent experiments, n > 50 cells per condition. Statistical significance was determined by two-sided, unpaired t test. ****, 0.0001 > P. (H) Images of WGA labeled retraction fibers and migrasomes in 231-WT (upper) and 231-PD-L1KO cells (bottom). Scale bars: 10 μm. Red dotted lines outline cell body. (I) Quantification of migrasome numbers/retraction fiber length. Data are shown as means ± SD from three independent experiments, n > 50 cells per condition. Statistical significance was determined by two-sided, unpaired t test. ****, 0.0001 > P.

PD-L1 concentrates at the cell rear and promotes rear retraction generating PD-L1 containing retraction fibers and migrasomes. (A) Left: Representative time-lapse microscopy images of PD-L1KO-PD-L1-EGFP MDA-MB-231 cells show PD-L1 expression during the formation of retraction fibers and migrasomes. Scale bar: 10 μm. Right: Quantification of PD-L1 signal from the rear toward inner cell body over time. Arrowheads indicate retracting cell rear. (B) Representative time-lapse microscopy images of 4T1-PD-L1-EGFP cells show PD-L1 localizes at retraction fibers and migrasomes. Scale bar: 10 μm. (C) Representative time-lapse microscopy images of PD-L1-EGFP PC3 cells show PD-L1 localizes at retraction fibers and migrasomes. Scale bar: 10 μm. (D) A representative image of WT MDA-MB-231 cells stained with F-actin (phalloidin, green) and PD-L1 (red). Co-localization pixels of PD-L1 and F-actin are shown in the black-white image on the bottom right. Scale bar: 10 μm. (E) Images of PD-L1-EGFP (green) and WGA (red) in migrating cells. The boxed region outlines retraction fibers and migrasomes. Single-channel images are shown as black-white images. Scale bar: 10 μm. (F) Representative time-lapse microscopy images of WT (top) or PD-L1KO (bottom) MDA-MB-231 cells pre-labeled with SiR-Actin. Scale bar: 10 μm. Arrowheads point to cell rear and retraction fibers. (G) Quantification of retraction fiber length from images of WT or PD-L1KO MDA-MB-231 and 4T1 cells. Data are shown as means ± SD from three independent experiments, n > 50 cells per condition. Statistical significance was determined by two-sided, unpaired t test. ****, 0.0001 > P. (H) Images of WGA labeled retraction fibers and migrasomes in 231-WT (upper) and 231-PD-L1KO cells (bottom). Scale bars: 10 μm. Red dotted lines outline cell body. (I) Quantification of migrasome numbers/retraction fiber length. Data are shown as means ± SD from three independent experiments, n > 50 cells per condition. Statistical significance was determined by two-sided, unpaired t test. ****, 0.0001 > P.

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