POT1 depletion does not induce significant levels of chromosome end fusions. (A) Metaphase spreads of the indicated HeLa cells with telomeric DNA detected by FISH (green). HeLa cells stably expressing the indicated shRNAs were infected with adenovirus expressing the TRF2 dominant-negative allele (AdTRF2^ΔBΔM) or a control adenovirus (Adβgal), and processed for chromosome analysis after 2 days. M indicates a marker chromosome with internal TTAGGG repeats. The inset shows an enlarged image of one chromosome-type fusion from the AdTRF2^ΔBΔM/POT1 shRNA panel. (B) Summary of the frequency of chromosome fusions in HeLa cells and HCT75 cells stably expressing POT1 shRNA and treated with the AdTRF2^ΔBΔM adenovirus to inhibit TRF2. Chromosomes were analyzed 2 days after introduction of AdTRF2^ΔBΔM and 15 days after knockdown of POT1.

Differential effect of POT1 shRNA on proliferation of primary and transformed cells. (A) Graph showing growth curves of HeLa cells with and without POT1 shRNA. The inset shows the growth rate (PD/day) of these and additional HeLa cells with lowered POT1 level. (B) Graph showing proliferation of HeLa cells transfected with POT1 siRNA or control siRNA. (C) FACS profiles of BrdU-labeled HeLa cells infected with the indicated shRNA viruses. Insets show the % cells in G1, S, and G2/M. (D) Phase-contrast microscopic images of IMR90 cells 7 days after infection and selection with the indicated shRNA viruses stained for SA-β-galactosidase activity (Dimri et al, 1995) for 10 h. (E) Graph showing the effect of POT1 shRNA ex18 on the proliferation of IMR90 cells. The inset shows the growth rates (PD/day) in cells infected with the indicated shRNAs and a vector expressing a POT1 cDNA resistant to ex18 shRNA (POT1^*). (F) Graph showing the effect of POT1 shRNAs on IMR90 cells transformed by introduction of SV40 large T antigen (pBabeNeoLT). The inset lists the growth rate of the cells.

POT1 determines the sequence at the 5′ end of human chromosomes. (A) Schematic of the ends of human chromosomes and the 5′ telorette assay. The six telorettes and the 5′ ends to which they can ligate are shown. PCR primers used for amplification are shown schematically. (B–F) Products of the 5′ telorette assay using the indicated cell lines. Each telorette was used for 2–5 independent assays and the products were run in separate lanes. The sequences of the 5′ end detected with each telorette is shown above the groups of lanes. POT1^* is a vector expressing full-length POT1 mutated to create resistance to the ex18 target site. The MWs of the detected products range from 1 to 8 kb.

POT1 and POT1–55 and their depletion with RNAi. (A) Schematic of two POT1 mRNAs and the proteins they encode. The immunoblot to the right shows HTC75 cells infected with the pLPC vector or the same vector expressing the POT1–55 form from V4 mRNA. Dark fill: OB folds. Light fill: TPP1 interacting domain. (B) Quantitative immunoblot to determine the level of POT1 knockdown in HelaS3 cells. Serial dilutions of vector protein extract were compared to the indicated relative cell equivalents of ex18 and ex8a knockdown cell extracts.

Transient telomere damage response upon POT1 depletion. (A) TIFs induced by POT1 shRNA ex18. BJ/hTERT cells were processed for TIF analysis 8 days after infection and selection of the ex18 vector or the vector control by IF for TRF1 (red) and 53BP1 (green) or γ-H2AX (green). Merged images are shown with DAPI. (B) Quantification of the induction of TIFs by POT1 ex18 shRNA. IF for TRF1 and 53BP1 (see (A)) was used and randomly selected groups of cells were evaluated for the number of TRF1 signals per cell that contained a 53BP1 signal. The bars show the percentage of cells containing 10 or more TIFs. (C) The majority of the telomeres in TIF-positive cells colocalize with 53BP1. TIF-positive BJ (•) and BJ/hTERT (○) cells were selected and imaged using deconvolution software. Each point in the graph represents one TIF-positive cell and shows the number of TRF1 signals plotted versus the number of TRF1 signals containing 53BP1. Points above the line represent cells in which more than 50% of the TRF1 signals contained 53BP1. (D) Transient TIFs in G1. HeLa cells expressing POT1 shRNA ex18 or vector control were released from double-thymidine block and processed for IF at the indicated time points. FACS profiles are shown below the IF images. Top: 53BP1 signal (green) merged with DAPI (blue). Bottom: 53BP1, TRF1 (red), and DAPI signals merged. (E) Quantification of cell cycle dependence of TIF-positive cells. Quantification as in (B), using the cells shown in (D).

POT1 is required for the maintenance of the 3′ overhang. (A, C, F) In-gel assay for ss TTAGGG repeats with the indicated cell lines expressing the indicated shRNAs. DNA was isolated 7–10 days post-infection, cut with MboI and AluI and processed by in-gel hybridization to a (CCCTAA)4 probe to detect ss TTAGGG repeats (left panels). The DNA was subsequently denatured in situ and rehybridized to the probe to detect the total TTAGGG repeat signal (right panels). (B, D, G) Bar graphs representing quantified overhang signals. Overhang signals in each lane were normalized to the total TTAGGG signal. The values are expressed relative to the value obtained with mock or vector (B) infected cells. The values in (B) represent averages of three experiments (SDs indicated). Below the bargraph, + and − indicate whether the shRNA targets POT1 and/or POT1–55. (E, H) Immunoblots of POT1 and POT1–55 levels in BJ (E) and BJ/hTERT (H) cells infected with retroviruses expressing the indicated shRNAs.