Effect of expression of GSE24-2 on telomerase activity in response to cisplatin. (A) pLNCX and GSE24-2 cells were treated with 3 μg/mL c-DDP for the indicated time periods. Telomerase activity was detected with the TRAP assay kit. Different extract dilutions are presented for each TRAP assay (total protein amount indicated in triangles). Asterisks indicate the samples that showed important differences and square brackets indicate the important bands. The experiments were repeated 3 times, with similar results. (B) Telomerase activity following in vitro exposure to platinum. GSE24-2 and pLNCX cell extracts were exposed in vitro to platinum with the indicated doses of cisplatin (0-30 μg/mL). Telomerase activity was detected as in panel A, using a telomere-specific template. The experiments were repeated 3 times, with similar results. (C) Telomerase activity following in vitro treatment of 293T extracts with cisplatin and the 24-2 peptide. After in vitro treatment of 293T extracts with cisplatin, different amounts of the 24-2 purified peptide (lanes 3-5), heat-inactivated 24-2 peptide (lane 6), or control peptide (lane 7) were added to the TRAP assay mix. A telomere-specific template was used for the telomerase assay. The experiment was repeated 3 times, with similar results. (D) Band-shift assay of cell extracts expressing GSE24-2. Nuclear cell extracts of cells expressing the plasmid pcDNA3–9E1024–2 or pcDNA3–9E10 were subjected to a band-shift retardation assay. A TEL1 probe containing a telomere-specific sequence was used. Binding specificity was established using 9E10 specific antibody (lane 3) and the CXext oligonucleotide that hybridized to the TEL1 overhang as a competitor (lanes 4-5). The experiment was repeated 3 times, with similar results.