Fig 1.
Phase-contrast images of αB-crystallin-overexpressing and knockdown L6 myoblasts, and C6 glioma cells.
Compared with wild-type cells, αB-crystallin-overexpressing cells had a more spread shape and knockdown cells showed a narrow, fibroblast-like shape in both cell types.
Fig 2.
Cell morphology and αB-crystallin level.
(A) Characteristic cell morphology is specific to αB-crystallin level as revealed by inhibitory antibody injection and (B) injection of purified bovine lens protein αB-crystallin into wild-type, αB-crystallin-overexpressing and knockdown C6 cells. Asterisk indicates P value of <0.05.
Fig 3.
Morphology-dependent cell proliferation study of αB-crystallin-overexpressing cells and knockdown C6 cells.
See detail in the text. Bar = 40 μm in A.
Fig 4.
αB-crystallin-overexpressing cells and knockdown C6 cells: shape change after Nocodazole treatment.
Images from just after the drug addition (final 10 μg/mL; 0 min, left panel) and after 90 min (right panel) (A). Bar = 40 μm. Cell area (B) and Shape index (C) change of C6 cells after Nocodazole treatment. N = 20.
Fig 5.
Effect of paclitaxel on αB-crystallin-overexpressing C6 cells and knockdown cells.
Cell shape change after paclitaxel treatment. Just after the addition of drug (final 20 μM; 0 min, left panel) and 90 min later (right panel) (A). Cell area (B) and Shape index (C) change of C6 cells after paclitaxel treatment (final 20 μM). N = 20.
Fig 6.
Effect of cytochalasin D on C6 αB-crystallin-overexpressing and knockdown cells.
Just after drug addition (final 0.4 μM; 0 min, left panel) and 45 min (right panel) (A). Cell area (B) and Shape index (C) change of C6 cells after Cytochalasin D (final 0.4 μM). N = 20.
Fig 7.
Impact of Rac1 on morphology of αB-crystallin-overexpressing and knockdown cells.
C6 cells were transfected with EGFP-Rac1 (wild-type), EGFP-constitutively active Rac1 (V12), and EGFP-dominant-negative Rac1 (N17), and fluorescence images were taken after 12 h. αB-crystallin knockdown C6 cells that expressed Rac 1V12 showed enlarged peripheral ruffles at both ends of spindle-shaped cells. On the other hand, complete loss of lengthened pseudopods was observed in Rac1N17 transfected cells (A). Area change (B) and shape index (C) of EGFP-Rac1, Rac1V12, Rac1N17 expressed wild-type, αB-crystallin overexpressed, and knockdown C6 cells. ** = P<0.01, *P = 0.05, n = 50.
Fig 8.
αB-crystallin-overexpressing cells (left column), and knockdown (right column) L6 cells at 0 h, 1 h, and 2 h time points. Bar is 30 μm.
Fig 9.
Mode of migration of wild-type, αB-crystallin overexpressing and knockdown cells.
Bar is 50 μm. Time-lapse images were recorded for 2 h at 10 min intervals and traces of the migration were drawn on the right hand graph. Circles on the left panel indicate the center of the cells at the beginning of the image recordings.
Fig 10.
Time-dependent migration of wild-type, αB-crystallin-overexpressing and knockdown L6 myoblast cells (n = 200).
Cumulative distances moved were obtained from images captured every 10 min (A). Time-dependent migration of wild-type, αB-crystallin-overexpressing and knockdown C6 glioma cells (n = 300). Cumulative moving distances were obtained from images captured every 10 min (B).
Fig 11.
Focal adhesion-stress fiber phenotype is αB-crystallin dependent.
Wild-type, αB-crystallin overexpressed, and knockdown L6 cells were visualized for F-actin (green), vinculin (red), and nucleus (blue). Bar is 20 μm.
Fig 12.
Time-lapse imaging of αB-crystallin knockdown L6 cells.
Directional cell migration as fast as about 10 μm/ h are typically seen. Position of the nuclei are indicated by asterisks and arrows indicate the migratory direction. Bar is 20 μm (A). Asymmetric distribution of nucleus, F-actin stress fiber, and focal adhesion of αB-crystallin knockdown cells (B, inverted contrast image of Fig 11) are coincide with asymmetric polymerization of microtubule caused by αB-crystallin knockdown. Bar is 20 μm (C).
Fig 13.
Average migration speed of cells.
Fast migrating leukocytes (~300–1500 μm/hr.) [37]; single motile breast cancer cells (180 μm/hr) [38]; neuron migration in developing cerebral cortex (~60 μm/hr) [39]; normal human epidermal keratinocytes (58.2±2.4 μm/hr) [40]; migratory somatic cells (37±15 μm/hr) [41]; αB-crystallin knockdown C6 glioma cells (40.1 μm/hr, this study); αB-crystallin knockdown L6 myoblast cells (20.2 μm/hr, this study); mouse E9.0 primordial germ cells (16.2±2.5 μm/hr) [42]; primary human dermal fibroblasts (15 μm/hr) [43]; astrocytes (15 μm/hr) [44]; primary human myoblasts (10.5±5.8 μm/hr) [45]; C6 glioma cell (12.1 μm/hr, this study); L6 myoblast cells (7.2 μm/hr, this study); αB-crystallin-overexpressing C6 glioma cells (8.5 μm/hr, this study); αB-crystallin-overexpressing L6 myoblast cells (5.8 μm/hr, this study).
Table 1.
αB-crystallin-dependent cell characteristics in rat C6 glioma cells.
Table 2.
αB-crystallin dependent cell characteristics in rat L6 myoblast cells.