FIGURE 4:

Imetelstat inhibits telomerase in vivo but does not affect RNP assembly. (A) Western blot of telomerase immunopurified from HeLa cells expressing Halo-TERT from the endogenous TERT locus, after 24-h treatment with 2 µM imetelstat or 2 µM mismatched control oligonucleotide, probed with an anti-TERT antibody. (B) Northern blot of RNA extracted from telomerase immunopurified from HeLa cells expressing Halo-TERT after 24-h treatment with 2 µM imetelstat, 2 µM mismatched control oligonucleotide, or no drug. In vitro–transcribed full-length (FL) TR was included as size standard and truncated TR 34-328 as loading and recovery control. Blots were probed with three radiolabeled oligonucleotides antisense to TR. (C) Direct telomerase assays at 150 mM KCl of telomerase immuno-purified from HeLa cells expressing Halo-TERT from the endogenous TERT locus, after 24-h treatment with 2 µM imetelstat, 2 µM mismatched control oligonucleotide, or no drug; each concentration in duplicate. LC1, LC2, and LC3, labeled DNA loading controls. (D) Quantification of the activity of telomerase purified from HeLa cells expressing Halo-TERT from the endogenous TERT locus, after 24-h treatment with 2 µM imetelstat or 2 µM mismatched control oligonucleotide, normalized to No Drug and loading control (n = 4, mean).