Figure 3

Down-regulation of CD98hc results in reduced migration, proliferation and survival in Caki2 cells. (A) Cell adhesion: Low and highCD98hc/Caki2 cells were seeded for 10, 20, 40 and 60 min on 10 μg/ml fibronectin and stained with crystal violet. Cells were counted per field with 20x magnification (n= 3, data represent the mean ± SEM, p=0.753), (1 field = 0.5 mm²). (B) Cell spreading: After attachments to 10 μg/ml fibronectin spreading was measured after 10, 20, 40 and 60 min. n=3, data represent the mean ± SEM, *p<0.001 (C) Cell transmigration: cell transmigration was analyzed using a modified Boyden chamber system, which revealed diminished migratory capability in lowCD98hc/Caki2 cells (white bars: mean 20 ± 3 cells/field after four hours, mean 122 ± 46 cells/field after 12 h and 520 ± 67 cells/field after 24 h) when compared to highCD98hc/Caki2 cells (black bars: mean 103 ± 5 cells/field after four hours, mean 495 ± 89 cells/field after 12 h and 1257 ± 346 cells/field 0.5 mm²) (D) Cell proliferation: cell [³ H] thymidine incorporation was reduced to 52 ± 3% after 24 h incubation under normal medium conditions in lowCD98hc/Caki2 cells when compared to control (highCD98hc/Caki2). n = 3, data represent the mean ± SEM, * p < 0.001. (E) Cell survival: Apoptosis induced by serum starvation or anoikis was measured via Annexin V/PI – FACS analysis, which revealed that lowCD98hc/Caki2 cells were more prone to apoptosis. Upper left panels point out necrotic cells. Representative dot blots given; n=5.