FIGURE 8.
Ectopically expressing a kinase-dead form of FAK in MVs derived from transformed MEFs reduces their cell survival-promoting capabilities. A, MEFs induced to express onco-Dbl were mock-transfected or transfected with HA-tagged forms of either FAK WT or FAK KD and lysed. The MVs generated by these cells were also collected, and a portion of them was directly lysed, whereas the rest of the isolated MVs were added to cultures of NIH-3T3 fibroblasts for 2 h before the cells were lysed. Western blotting analysis using HA, P-FAKTyr397, FAK, and actin antibodies was performed on the transfected MEFs (Donor MEFs), the MVs the cells generated (MVs from Donor MEFs), as well as on NIH-3T3 cells that were treated without (no MVs added) or with the various MV preparations (Recipient Fibroblasts). B, NIH-3T3 cells were cultured in serum-free medium supplemented without (Serum Starved) or with MVs collected from onco-Dbl-transformed MEFs that had been mock-transfected or transfected with HA-tagged forms of either FAK WT or FAK KD. Approximately 48 h later, the cells were stained with DAPI to identify the condensed/blebbed (apoptotic) nuclei. C, Western blotting analysis using HA, FAK, and actin antibodies was performed on NIH-3T3 cells that had been mock-transfected or transfected with either HA-tagged forms of FAK WT or FAK KD. D, NIH-3T3 cells that had been mock-transfected or were ectopically expressing HA-tagged FAK WT or HA-tagged FAK KD were serum-starved for ∼48 h, at which point the cells were stained with an HA antibody to identify the transfectants and DAPI to identify condensed/blebbed (apoptotic) nuclei. The experiments in A–D were performed three separate times, each time yielding similar results. Molecular weight markers (in kilodaltons) are included along the right side of each blot in A and C. The data shown in B and D represent the mean ± S.D. Student's t tests were performed. *, p < 0.05; ***, p < 0.001; n.s., not significant.