Figure 3.
PD-L1 and the β4 integrin interact physically and functionally at cell rear. (A) Representative images of PD-L1KO-PD-L1-EGFP MDA-MB-231 cells plated on laminin-332-coated coverslips show PD-L1 distribution. Scale bar: 10 μm. Arrowhead indicates migrasome containing retraction fibers. (B) Representative images of PD-L1KO-PD-L1-EGFP MDA-MB-231 cells plated on collagen-I coated coverslips show PD-L1 distribution. Scale bar: 10 μm. (C) Quantification of retraction fiber length from images of PD-L1KO-PD-L1-EGFP MDA-MB-231 cells plated on laminin-332 or collagen-I-coated coverslips. Data are shown as means ± SD from three independent experiments, n > 60 cells per condition. Statistical significance was determined by two-sided, unpaired t test. ****, 0.0001 > P. (D) An image of PD-L1KO-PD-L1-EGFP MDA-MB-231 cells stained with β4 integrin shows PD-L1 (green) and integrin β4 (red) distribution during cell migration. Scale bar: 10 μm. Arrowhead points to cell rear. (E) Representative images from PLA of PD-L1KO-PD-L1-EGFP MDA-MB-231 cells. Left: Negative control shows images without primary antibody treatment. Middle left: PLA results show PD-L1 protein localization (green), and β4 integrin and PD-L1 association (PLA: β4 PD-L1, red). Nucleus in blue. Middle: A higher magnification image from the boxed region in the left image shows that PD-L1 associates with β4 integrin at the tips and intersections of retraction fibers. Middle right: PLA results show PD-L1 protein localization (green), and integrin β1 and PD-L1 association (PLA: β1 PD-L1, red). Right: PLA results show α3 integrin protein localization (green) and α3 integrin and β1 integrin association (PLA: α3 β1, red). Scale bar: 10 μm. (F) Quantification of the number of foci per cell from PLA assays mentioned above. Data are shown as means ± SD from three independent experiments, n > 35 cells per condition. Statistical significance was determined by two-sided, unpaired t test.  *, 0.05 > P ≥ 0.01; ****, 0.0001 > P. (G) Representative time-lapse microscopy images of 4T1-β4-tdTomato-PD-L1-EGFP cells show PD-L1 (green) and β4 integrin (red) distribution during the formation of retraction fibers. Scale bar: 10 μm. Arrowheads point to regions of retraction “roots” and retraction fibers. (H) Left: a representative image of PC3-PD-L1-EGFP cells shows localization of β4 integrin (β4, red) and PD-L1 (green). Right: A higher magnification image from the boxed region in the left image show co-localization of β4 integrin (β4, red) and PD-L1 (green) in retraction fibers. Scale bar: 10 μm. (I) Quantification of length of retraction fibers in WT or PD-L1KO MDA-MB-231 cells when glass-bottomed dishes were coated with different concentrations of laminin-332 (LM332). Data are shown as means ± SD (n = 50 cells per condition, three independent experiments). Statistical significance was determined by two-sided, unpaired t test. *, 0.05 > P ≥ 0.01; **, 0.01 > P ≥ 0.001; ***, 0.001 > P ≥ 0.0001. (J) Quantification of numbers of migrasomes/length of retraction fibers in WT or PD-L1KO MDA-MB-231 cells when glass-bottomed dishes were coated with different concentrations of laminin-332 (LM332). Data are shown as means ± SD (n = 50 cells per condition, three independent experiments). Statistical significance was determined by two-sided, unpaired t test. **, 0.01 > P ≥ 0.001; **** 0.0001 > P.

PD-L1 and the β4 integrin interact physically and functionally at cell rear. (A) Representative images of PD-L1KO-PD-L1-EGFP MDA-MB-231 cells plated on laminin-332-coated coverslips show PD-L1 distribution. Scale bar: 10 μm. Arrowhead indicates migrasome containing retraction fibers. (B) Representative images of PD-L1KO-PD-L1-EGFP MDA-MB-231 cells plated on collagen-I coated coverslips show PD-L1 distribution. Scale bar: 10 μm. (C) Quantification of retraction fiber length from images of PD-L1KO-PD-L1-EGFP MDA-MB-231 cells plated on laminin-332 or collagen-I-coated coverslips. Data are shown as means ± SD from three independent experiments, n > 60 cells per condition. Statistical significance was determined by two-sided, unpaired t test. ****, 0.0001 > P. (D) An image of PD-L1KO-PD-L1-EGFP MDA-MB-231 cells stained with β4 integrin shows PD-L1 (green) and integrin β4 (red) distribution during cell migration. Scale bar: 10 μm. Arrowhead points to cell rear. (E) Representative images from PLA of PD-L1KO-PD-L1-EGFP MDA-MB-231 cells. Left: Negative control shows images without primary antibody treatment. Middle left: PLA results show PD-L1 protein localization (green), and β4 integrin and PD-L1 association (PLA: β4 PD-L1, red). Nucleus in blue. Middle: A higher magnification image from the boxed region in the left image shows that PD-L1 associates with β4 integrin at the tips and intersections of retraction fibers. Middle right: PLA results show PD-L1 protein localization (green), and integrin β1 and PD-L1 association (PLA: β1 PD-L1, red). Right: PLA results show α3 integrin protein localization (green) and α3 integrin and β1 integrin association (PLA: α3 β1, red). Scale bar: 10 μm. (F) Quantification of the number of foci per cell from PLA assays mentioned above. Data are shown as means ± SD from three independent experiments, n > 35 cells per condition. Statistical significance was determined by two-sided, unpaired t test.  *, 0.05 > P ≥ 0.01; ****, 0.0001 > P. (G) Representative time-lapse microscopy images of 4T1-β4-tdTomato-PD-L1-EGFP cells show PD-L1 (green) and β4 integrin (red) distribution during the formation of retraction fibers. Scale bar: 10 μm. Arrowheads point to regions of retraction “roots” and retraction fibers. (H) Left: a representative image of PC3-PD-L1-EGFP cells shows localization of β4 integrin (β4, red) and PD-L1 (green). Right: A higher magnification image from the boxed region in the left image show co-localization of β4 integrin (β4, red) and PD-L1 (green) in retraction fibers. Scale bar: 10 μm. (I) Quantification of length of retraction fibers in WT or PD-L1KO MDA-MB-231 cells when glass-bottomed dishes were coated with different concentrations of laminin-332 (LM332). Data are shown as means ± SD (n = 50 cells per condition, three independent experiments). Statistical significance was determined by two-sided, unpaired t test. *, 0.05 > P ≥ 0.01; **, 0.01 > P ≥ 0.001; ***, 0.001 > P ≥ 0.0001. (J) Quantification of numbers of migrasomes/length of retraction fibers in WT or PD-L1KO MDA-MB-231 cells when glass-bottomed dishes were coated with different concentrations of laminin-332 (LM332). Data are shown as means ± SD (n = 50 cells per condition, three independent experiments). Statistical significance was determined by two-sided, unpaired t test. **, 0.01 > P ≥ 0.001; **** 0.0001 > P.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal