Figure 5.
Figure 5. Pore size–dependent migration and deformation of the nucleus in mononuclear breast cancer cells, T-blasts, and PMN. (A) Lamin A/C content in distinct cell types. Lanes were loaded with whole-cell lysates normalized to GAPDH content and immunoblotted for lamin A/C. (B, D, and F) Migration efficiencies of the indicated cell types in rat tail collagen of different density in the absence or presence of GM6001. Steady-state speeds of single cells monitored over 24 h (MDA/MT1) or 2 h (T-blasts, PMNs). Dashed horizontal lines, subtotal (90% inhibition) and absolute (99% inhibition) migration limit (dashed top lane indicating 100% of MMP-independent migration). Box and whiskers show the medians, 25th/75th and 5th/95th percentiles (50–200 cells, 2–6 independent experiments). ***, P < 0.0001; n.s., not significant. (C, E, and G) Moving and immobilized phenotypes of MDA/MT1 cells, T-blasts, and PMN in the presence of GM6001 in rat tail collagen of different pore sizes (numbers, collagen concentration in mg/ml). Cultures were fixed at the end-point (16 h, MDA/MT1; 2 h, leukocytes) and stained as indicated. Insets, DAPI. Arrows, direction of migration and protrusion, respectively. Tables (C and E), ranges of nuclear cross sections and diameters and their frequencies at different collagen density (numbers in percent, full dataset shown in Fig. S4, C and D) associated with intact or abrogated migration (17–30 independent cells). Insets (G), schematics of different nuclear shapes, including rounded (immobilized), fully or partially unfolded. (H) Change of nuclear morphology in migrating PMN (rat tail collagen, 1.7 mg/ml) over 600-s time period (full sequence shown in Fig. S4 E, example 1; and Video 8). Insets, DAPI signal (left) and schematics of nuclear shape (right). Arrowhead, saltatory migration phase. (I) Nuclear elongation index (top graph) and distance between nucleus and leading extension (pseudopod–nucleus distance) plotted over time. Measurement as indicated by black arrows. Graphs show one representative example of a moving cell (red lines; compare to Fig. S4 E, example 2) and immobilized cell (black lines; compare to Fig. S4 F, example 1) out of n = 3. Bars: (C) 10 µm; (E, G, and H) 5 µm.

Pore size–dependent migration and deformation of the nucleus in mononuclear breast cancer cells, T-blasts, and PMN. (A) Lamin A/C content in distinct cell types. Lanes were loaded with whole-cell lysates normalized to GAPDH content and immunoblotted for lamin A/C. (B, D, and F) Migration efficiencies of the indicated cell types in rat tail collagen of different density in the absence or presence of GM6001. Steady-state speeds of single cells monitored over 24 h (MDA/MT1) or 2 h (T-blasts, PMNs). Dashed horizontal lines, subtotal (90% inhibition) and absolute (99% inhibition) migration limit (dashed top lane indicating 100% of MMP-independent migration). Box and whiskers show the medians, 25th/75th and 5th/95th percentiles (50–200 cells, 2–6 independent experiments). ***, P < 0.0001; n.s., not significant. (C, E, and G) Moving and immobilized phenotypes of MDA/MT1 cells, T-blasts, and PMN in the presence of GM6001 in rat tail collagen of different pore sizes (numbers, collagen concentration in mg/ml). Cultures were fixed at the end-point (16 h, MDA/MT1; 2 h, leukocytes) and stained as indicated. Insets, DAPI. Arrows, direction of migration and protrusion, respectively. Tables (C and E), ranges of nuclear cross sections and diameters and their frequencies at different collagen density (numbers in percent, full dataset shown in Fig. S4, C and D) associated with intact or abrogated migration (17–30 independent cells). Insets (G), schematics of different nuclear shapes, including rounded (immobilized), fully or partially unfolded. (H) Change of nuclear morphology in migrating PMN (rat tail collagen, 1.7 mg/ml) over 600-s time period (full sequence shown in Fig. S4 E, example 1; and Video 8). Insets, DAPI signal (left) and schematics of nuclear shape (right). Arrowhead, saltatory migration phase. (I) Nuclear elongation index (top graph) and distance between nucleus and leading extension (pseudopod–nucleus distance) plotted over time. Measurement as indicated by black arrows. Graphs show one representative example of a moving cell (red lines; compare to Fig. S4 E, example 2) and immobilized cell (black lines; compare to Fig. S4 F, example 1) out of n = 3. Bars: (C) 10 µm; (E, G, and H) 5 µm.

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