Functional consequences of reintroducing EPHB4 expression into Raji cells. (A) Lentiviral constructs for transducing EPHB4 and controls. (B) Ectopic expression and membrane localization of lentivirus-delivered EPHB4 in Raji cells. Raji cells were infected with FUGW-hEphB4 or FUGW-lentivirus. Cells were examined by fluorescence microscopy (i, ×40) or phase contrast (ii, ×40). Raji cells transduced with FUGW- hEphB4 resulted in the expression of EPHB4 as shown by immunofluorescence staining with Alexa 594 (red) on the cell membrane (iii, ×60), whereas the empty vector had no effect and had only shown 4′,6-diamidino-2-phenylindole nuclear staining (iv, ×60). For more information see “Immunofluorescence microscopy.” (C) Western blot analysis of EPHB4 expression in Raji and CEM cells transduced with EphB4 lentivirus or empty vector. (D) Assessment of GFP positive Raji cell proliferation after infected with FUGW-EphB4 lentivirus or control using flow cytometric GFP sorting. Growth curves for GFP-positive Raji cells were determined for each of 3 replicates averaged at the indicated time points. (E) Analysis of apoptosis in Raji cells 2 days after lentivirus infection using flow cytometry and annexin V–PE staining. (F) FACS analysis of cell-cycle distributions measured 3 days after lentivirus infection. The percentage of cells in sub-G1 is presented. (G) For colony formation assay, Raji cells infected with FUGW-EphB4 lentivirus or controls were grown in soft agarose. Bar graph (bottom) represents experiments shown on top. **P < .01.