Fig. 2.

T-oligos with a blocked 3′ end are inactive. (A) Cultures of human MM-AN melanoma cells were treated once at time 0 with either 40 μM T-oligo (GTTAGGGTTAG) with phosphodiester linkages or with this oligonucleotide synthesized with phosphorothioate linkages (PS-T-oligo) to the control, complementary oligonucleotide, or diluent alone and harvested after 4 days. Apoptotic cells were identified as the sub-G₀/G₁ cells in FACS analysis. All experimental conditions were done in triplicate. The averages and SDs shown are calculated from two combined experiments. (B) Normal newborn fibroblasts were treated once at time 0 with either diluent alone or 40 μM of the following T-oligos: lane 1, all phosphodiester (no phosphorothioate) linkages (GTTAGGGTTAG); lane 2, two phosphorothioate linkages at both the 3′ and the 5′ ends (G_sT_sTAGGGTT_sA_sG); lane 3, all phosphorothioate linkages (G_sT_sT_sA_sG_sG_sG_sT_sT_sA_sG); lane 4, just the 5′ end blocked with 2 phosphorotioate linkages (G_sT_sTAGGGTTAG); or lane 5, just the 3′ end blocked with 2 phosphorothioate linkages (GTTAGGGTT_sA_sG). After 48 h, the cells were collected, and the proteins were analyzed by Western blot. Fibroblasts treated with 10 Gy ionizing radiation (IR+) or sham irradiated (IR−) were included as positive and negative controls.