Figure 5

Reconstitution of CD98hc rescues the rates of migration, proliferation and survival in low CD98hc CaKi2 cells. (A) Cell adhesion in reconstitution mutant expressing CaKi2 cells: Adhesion was slightly, but not significant reduced in trunsilCD98hc Caki2 cells. silCD98hc expression or poinsilCD98hc expression showed no relevant correlation in cell adhesion. (n = 4, data represent the mean ± SEM, p > 0.05). (B) Spreading: Reconstitution with silCD98hc rescued the low CD98hc cell spreading phenotype starting after 20 min. However, the deletion mutant trunsilCD98hc Caki2 cells could not rescue reduced spreading at any time point, but had a similar spreading behavior as lowCD98hc/Caki2 cells. poinsilCD98hc/Caki2 cells had a similar rescued spreading effect as silCD98hc reconstitution in Caki2 cells. n = 3, data represent the mean ± SEM, * p < 0.001 (C) Transmigration of cells expressing reconstitution mutants: Transmigration in a modified Boyden chamber system revealed diminished migratory capability in trunsilCD98hc/Caki2 cells (mean 13 ± 2 cells/field after 4 h), compared to silCD98hc Caki2 cells (mean 100 ± 7 cells/field after 4 h) as well as to poinsilCD98hc Caki2 cells (mean 99 ± 3 cells/field after 4 h). n = 3, mean ± SEM, * p < 0.001 (D) Cell proliferation in reconstitution mutants: Reconstitution of silCD98hc was able to rescue the proliferative phenotype of low CD98hc expressing cells. trunsilCD98hc/Caki2 cells had similar proliferative activity such as lowCD98hc/Caki2 cells, whereas poinsilCD98hc/Caki2 cells could partially reconstitute for cell proliferation, however, the lower proliferative activity compared to highCD98hc/Caki2 was statistical not significant. n = 4, mean ± SEM, * p < 0.001, # > 0.05 (E) Survival in reconstitution mutant expressing cells: Apoptosis assay measured via Annexin V/PI– FACS analysis. Upper left panels reflect necrotic cells. Upper left panels reveal necrotic cells. Representative dot blots given; n=3.