(A) MDA-MB-231 human breast cancer cells were transfected for 72 hours with 100 nM 4E-ASO4 or mismatch control. Representative photomicrographs illustrate the dramatic increase in TUNEL staining after 4E-ASO4 transfection. Nuclear staining is revealed by Hoechst dye. (B) The mean percentage of cells positive for TUNEL or activated caspase-3 is depicted for both MDA-MB-231 and H460 non–small cell lung cancer cells (± SEM). Data in A and B are representative of more than 4 separate determinations. (C) HUVECs were plated on Matrigel and transfected with the 4E-ASO4, 4E-ASO2, or non-silencing ASO controls as indicated (ASO ctrlE, 5′-TGTTACAGTCTTGTACCCTT-3′ and "randomer" ASO ctrlF, a random mix of 420 possible 20-mer nucleotide sequences). The formation of vessel-like tubes was scored semi-quantitatively on a scale of 1 to 5. Data are presented as the mean tube score from 4 separate determinations ± SD. (D) Mean eIF4E expression ± SEM was evaluated from parallel plates by quantitative RT-PCR for the controls and the 4E-ASO4–transfected HUVECs. RT-PCR for the cells treated with 4E-ASO2 was not performed. (E) HUVECs were transfected for 48 hours with 150 nM 4E-ASO4 or mismatch control ASO, replated on dermal fibroblasts, and incubated for 6 days to assess the formation of chord-like structures. Endothelial cells were visualized immunohistochemically with an anti-CD31 antibody (green). Hoechst stain reveals the nuclei of the dermal fibroblasts (blue). Western blot analyses of eIF4E expression 48 hours posttransfection are shown. Data are representative of 5 separate experiments.