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. 2005 Apr 27;25(17):4406–4419. doi: 10.1523/JNEUROSCI.4955-04.2005

Figure 6.

Figure 6.

Trans-well cell migration assay. A, Effects of plasmid transfection on trans-well migration of PC12 cells. PC12 cells were transfected with a mock vector plus pEGFP-N2 (lanes 1, 2), pEGFP-FL-P-Rex1 (lanes 3, 4), pEGFP-ΔDH-P-Rex1 (lane 5, 6), or pcDNA3-Myc-N17Rac1 plus pEGFP-N2 (lanes 7, 8). Cell suspensions were applied to the upper wells of the trans-well chambers (modified Boyden chambers) (Ho et al., 2001), and GFP-positive cells that migrated to the bottom side of the filter were counted after 16-19 h of incubation with or without NGF in the lower wells. The relative migration score represents the relative percentage of cell migration compared with that of mock-transfected PC12 cells with NGF treatment (lane 2) (see Materials and Methods). B, The ability of P-Rex1 to enhance PC12 cell motility with NGF treatment is dependent on Rac activity. pEGFP-FL-P-Rex1 was cotransfected with a mock vector (lanes 1, 2) or pcDNA3-Myc-N17Rac1 (lanes 3, 4) in PC12 cells, which were subsequently subjected to the trans-well migration assay with or without NGF in the bottom chambers. The relative migration score represents the relative percentage of the extent of cell migration compared with that of the control experiment (lane 2). The amounts of pEGFP-FL-P-Rex1 plasmid used were one-half of that used in A, resulting in a slightly lower response in FL-P-Rex1-expressing cells in B, lanes 1 and 2, than in A, lanes 3 and 4. C, RNA interference for P-Rex1 resulted in suppression of NGF-induced trans-well migration of PC12 cells. a, pEGFP-N2 was cotransfected with a luciferase siRNA (lanes 1,2) or P-Rex1 siRNA (lanes 3, 4) into PC12 cells. After 4 d of incubation at 37°C, trans-well migration assays were performed for 4-5 h. The relative migration score represents the relative percentage of the extent of cell migration compared with that of the control experiment (lane 2). b, Western blotting analysis showed downregulation of endogenous P-Rex1 protein. All assays were repeated at least three times. Data were evaluated by using Student's t test. **p < 0.01.