Figure 7.
PD-L1 promotes rear contractility by enabling the β4 integrin to engage the cytoskeleton and activate RhoA. (A) Fluorescent images of WT (left) or PD-L1KO (right) MDA-MB-231 cells treated with 0.5% Triton X in PBS before fixation show distribution of cytoskeleton-associated integrin β4 (CSK:β4, red), and F-actin (green). Single channel only images are shown in black-white images. Scale bar: 10 μm. (B) Fluorescent images of PD-L1KO-PD-L1-EGFP (left) or PD-L1KO-PD-L1C272A-EGFP (right) MDA-MB-231 cells treated with 0.5% Triton X in PBS before fixation show distribution of cytoskeleton associated integrin β4 (CSK:β4). Scale bar: 10 μm. (C) Fluorescent images of live WT MDA-MB-231 cells expressing the plasmid of EGFP-AHPH (RhoA Biosensor) show distribution of integrin β4 (red) and activate RhoA (EGFP-AHPH, green). Single-channel only images are shown in black-white images. Arrowheads indicate cell rear. Scale bar: 10 μm. (D) Fluorescent images of live PD-L1KO MDA-MB-231 cells expressing the plasmid of EGFP-AHPH (RhoA Biosensor) show distribution of integrin β4 (red) and activate RhoA (EGFP-AHPH, green). Single channel only images are shown in black-white images. Scale bar: 10 μm. (E) Fluorescent images of live MDA-MB-231 cells treated with siCtrl (left) or siRNAs against β4 integrin (middle and right) show distribution of F-actin (red) and activate RhoA (EGFP-AHPH, green). Single channel only images are shown in black-white images. Arrowheads indicate cell rear. Scale bar: 10 μm. (F) Raichu-RhoA FRET ratio images of WT and PD-L1KO MDA-MB-231 cells. Scale bar: 10 μm. (G) Quantification of average rear Raichu-RhoA FRET ratio in WT and PD-L1KO MDA-MB-231 cells. Data are shown as box-whisker plots (5-95 percentile) from three independent experiments, n > 30 cells per condition. Statistical significance was determined by two-sided, unpaired t test. ****, 0.0001 > P. (H) Quantification of average rear Raichu-RhoA FRET ratio in MDA-MB-231 cells treated with siCtrl or siRNAs against β4 integrin. Data are shown as box-whisker plots (5-95 percentile) from three independent experiments, n > 30 cells per condition. Statistical significance was determined by two-sided, unpaired t test. **, 0.01 > P ≥ 0.001; ***, 0.001 > P ≥ 0.0001. (I) Fluorescent images of WT (top) or PD-L1KO (bottom) MDA-MB-231 cells show distribution of actin (phalloidin, green), and pMLC(S19) (red). Single channel only images are shown in black-white images. White arrowhead points to the cell rear. Scale bar: 10 μm. (J) Quantification of pMLC(S19) signal at cell rear and cell front in WT or PD-L1KO MDA-MB-231 cells. Data are shown as before-after plots from three independent experiments, n > 30 cells per condition. Statistical significance was determined by two-sided, paired t test.  ****, 0.0001 > P. (K) Fluorescent images of MDA-MB-231 cells treated with either H20 (WT-Ctrl, left) or Y27632 (10 µM for 1 h; middle) were compared with PD-L1KO cells (right) for the distribution of actin (phalloidin, red), and β4 integrin (green). White arrowhead points to elongated tails. Scale bar: 10 μm. (L) Quantification of the percentage of MDA-MB-231 cells described in K with elongated tails (left) and the length of their retraction fibers (right). Data are shown as means ± SD from three independent experiments, n > 30 cells per condition. Statistical significance was determined by two-sided, unpaired t test. **, 0.01 > P ≥ 0.001; ***, 0.001 > P ≥ 0.0001; ****, 0.0001 > P.

PD-L1 promotes rear contractility by enabling the β4 integrin to engage the cytoskeleton and activate RhoA. (A) Fluorescent images of WT (left) or PD-L1KO (right) MDA-MB-231 cells treated with 0.5% Triton X in PBS before fixation show distribution of cytoskeleton-associated integrin β4 (CSK:β4, red), and F-actin (green). Single channel only images are shown in black-white images. Scale bar: 10 μm. (B) Fluorescent images of PD-L1KO-PD-L1-EGFP (left) or PD-L1KO-PD-L1C272A-EGFP (right) MDA-MB-231 cells treated with 0.5% Triton X in PBS before fixation show distribution of cytoskeleton associated integrin β4 (CSK:β4). Scale bar: 10 μm. (C) Fluorescent images of live WT MDA-MB-231 cells expressing the plasmid of EGFP-AHPH (RhoA Biosensor) show distribution of integrin β4 (red) and activate RhoA (EGFP-AHPH, green). Single-channel only images are shown in black-white images. Arrowheads indicate cell rear. Scale bar: 10 μm. (D) Fluorescent images of live PD-L1KO MDA-MB-231 cells expressing the plasmid of EGFP-AHPH (RhoA Biosensor) show distribution of integrin β4 (red) and activate RhoA (EGFP-AHPH, green). Single channel only images are shown in black-white images. Scale bar: 10 μm. (E) Fluorescent images of live MDA-MB-231 cells treated with siCtrl (left) or siRNAs against β4 integrin (middle and right) show distribution of F-actin (red) and activate RhoA (EGFP-AHPH, green). Single channel only images are shown in black-white images. Arrowheads indicate cell rear. Scale bar: 10 μm. (F) Raichu-RhoA FRET ratio images of WT and PD-L1KO MDA-MB-231 cells. Scale bar: 10 μm. (G) Quantification of average rear Raichu-RhoA FRET ratio in WT and PD-L1KO MDA-MB-231 cells. Data are shown as box-whisker plots (5-95 percentile) from three independent experiments, n > 30 cells per condition. Statistical significance was determined by two-sided, unpaired t test. ****, 0.0001 > P. (H) Quantification of average rear Raichu-RhoA FRET ratio in MDA-MB-231 cells treated with siCtrl or siRNAs against β4 integrin. Data are shown as box-whisker plots (5-95 percentile) from three independent experiments, n > 30 cells per condition. Statistical significance was determined by two-sided, unpaired t test. **, 0.01 > P ≥ 0.001; ***, 0.001 > P ≥ 0.0001. (I) Fluorescent images of WT (top) or PD-L1KO (bottom) MDA-MB-231 cells show distribution of actin (phalloidin, green), and pMLC(S19) (red). Single channel only images are shown in black-white images. White arrowhead points to the cell rear. Scale bar: 10 μm. (J) Quantification of pMLC(S19) signal at cell rear and cell front in WT or PD-L1KO MDA-MB-231 cells. Data are shown as before-after plots from three independent experiments, n > 30 cells per condition. Statistical significance was determined by two-sided, paired t test.  ****, 0.0001 > P. (K) Fluorescent images of MDA-MB-231 cells treated with either H20 (WT-Ctrl, left) or Y27632 (10 µM for 1 h; middle) were compared with PD-L1KO cells (right) for the distribution of actin (phalloidin, red), and β4 integrin (green). White arrowhead points to elongated tails. Scale bar: 10 μm. (L) Quantification of the percentage of MDA-MB-231 cells described in K with elongated tails (left) and the length of their retraction fibers (right). Data are shown as means ± SD from three independent experiments, n > 30 cells per condition. Statistical significance was determined by two-sided, unpaired t test. **, 0.01 > P ≥ 0.001; ***, 0.001 > P ≥ 0.0001; ****, 0.0001 > P.

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