FIGURE 8.

Knockdown of TRF1 completely abolishes the ability of PinX1 to inhibit telomere elongation in human cells. A, establishment of stable HT1080 cell pools expressing PinX1 with or without TRF1 knockdown is shown. HT1080 stable cells expressing HA-PinX1 or control vector at PD 1 were infected with lentiviruses expressing TRF1 RNAi or vector control virus. After selection, levels of TRF1 in stable cell pools were determined by immunoblot with anti-TRF1 antibodies. B and C, TRF1 knockdown does not affect the ability of PinX1 to inhibit telomerase activity in human cells. Stable HT1080 cell pools that expressed HA-PinX1 (C) or control vector (B) and TRF1 RNAi or vector control virus were harvested at eight PDs, and telomerase-containing fractions were prepared followed by subjecting different amounts of proteins as indicated to the TRAP assay. Buffer alone or boiled lysates (B. Lys) were used in some assays as controls. Arrows point to the internal control (IC) for PCR amplification. D–F, TRF1 knockdown completely abolishes the ability of PinX1 to inhibit telomere elongation in human cells. Stable HT1080 cell pools that expressed HA-PinX1 (E) or control vector (D) and TRF1 RNAi or vector control virus were maintained continuously in culture and harvested at various PD as indicated followed by telomere genomic Southern blotting analysis (D and E). Average telomere restriction fragment length versus PD number was quantified using ImageQuant (F).