Epstein-Barr Virus-Encoded LMP1 Interacts with FGD4 to Activate Cdc42 and Thereby Promote Migration of Nasopharyngeal Carcinoma Cells
Figure 7
The action of the LMP1-FGD4-Cdc42 axis in NPC cells leads to actin rearrangement and increased cell motility.
(A) NPC-TW02 cells grown on coverslips were co-transfected with the indicated siRNA duplexes plus pEGFP-LMP1 or pEGFP vector, and then incubated for 48 h. Following a 6-h serum starvation, cells were fixed and stained with TRITC-conjugated phalloidin to examine actin filament (F-actin) arrangement. Images were acquired using a ZEISS LSM510 confocal microscope. Scale bar, 20 µm. (B) NPC-TW02 cells were co-transfected with the indicated siRNA duplexes and a Flag-LMP1 expression plasmid or empty vector. After 24 h, the cells were re-seeded for transwell migration assays as detailed in Materials and Methods. Images of migrating cells in each experiment were acquired at 200× magnification. The number of migrating cells was counted and presented as the mean ± SD of four independent experiments (*P<0.001, **P<0.01; paired t-test). (C) The level of active Cdc42 under each condition was determined by GST-CBD pull-down assay of cell lysates prepared from a portion of cells used in transwell migration assays, as described above. ND, not detectable. (D) NPC-TW02 cells were co-transfected with control or FGD4 siRNA plus a Flag-LMP1 expression plasmid or empty vector, with and without co-transfection of an expression plasmid for Flag-Cdc42DN (DN) or Flag-Cdc42CA (CA). At 24 h post-transfection, the cells were re-seeded for transwell migration assays. Images of migrating cells in each experiment were acquired at 200× magnification. The number of migrating cells was counted and presented as the mean ± SD of three independent experiments (*P<0.05, **P<0.01; paired t-test). (E) The level of active Cdc42 under each condition was determined by GST-CBD pull-down assay of cell lysates prepared from a portion of cells used in transwell migration assays, as described above.