Effects of suppressing hPOT1 on telomere dynamics. A. hPOT1 mRNA and protein levels. mRNA levels were determined by quantitative RT-PCR (top) and hPOT1 protein levels by immunoblotting (bottom) in BJ cells expressing either an hPOT1 shRNA (hPOT1sh #1) or a control shRNA (CONsh) in lentiviral constructs containing either puromycin (puro) or neomycin (neo) selection markers. Mean ± SD are shown for triplicate experiments. Arrows indicate hPOT1 isoforms. B. TRF Southern blotting of mortal BJ cells with passage. DNA was collected at the indicated population doublings (PD) after infection with CONsh (−) or hPOT1sh #1 (+). DNA size markers in kb are shown on the left. C. TRF Southern blotting of BJ cells that overexpress hTERT with successive passage. DNA was collected at the indicated PD after infection with a retrovirus encoding the hTERT cDNA in cells previously infected with CONsh (−) or hPOT1sh #1 (+). Lanes are numbered 1–12 for reference. D. Densitometry of selected lanes. The relative intensity at regular TRF length intervals is shown and mean TRF lengths for the entire lane are indicated. The upper left graph compares panel B lanes 1 (black diamonds) and 11 (grey squares); the upper middle graph compares panel B lanes 2 (black diamonds) and 12 (grey squares); the upper right graph compares panel B lanes 11 (black diamonds) and 12 (grey squares); the lower left graph compares panel C lanes 1 (black diamonds) and 11 (grey squares); the lower middle graph compares panel C lanes 1 (black diamonds) and 2 (grey squares); the lower right graph compares panel C lanes 11 (black diamonds) and 12 (grey squares).

Short-term effects of hPOT1 suppression in cells of different proliferative age. A. Doubling times of BJ and HFF cells. BJ or human foreskin fibroblast (HFF) cells infected with a control hairpin (CONsh) or one of two hairpins targeting hPOT1 (hPOT1sh #1 or #2) at the cumulative population doubling (PD) indicated. Doubling times were measured over 13–17 days in triplicate. B. hPOT1 mRNA levels in BJ and HFF cell at the indicated PD. Expression of hPOT1 for cell lines characterized in panel A using primers near the hPOT1sh #1 (top) or #2 (bottom) targeting site. C. SA β-gal staining. Values represent the percentage of cells that stained for SA β-gal. For these experiments, at least 100 total cells were scored in triplicate. D. TIF images. Staining for γ-H2AX (green) and telomeric DNA (red) are shown from two cells expressing hPOT1sh #1 that exhibited significant TIF formation, and two cells expressing CONsh that exhibited little to no TIF formation. E. Quantification of TIF formation. Values represent the percentage of nuclei containing >4 foci co-staining for γ-H2AX by indirect immunofluorescence and telomeric DNA by PNA FISH. At least 100 nuclei were scored in three independent experiments. F. DNA bridge formation. DAPI stained image of a representative DNA bridge (arrows) between two nuclei from PD 54 cells expressing hPOT1sh #1. DNA bridges were identified in 4% (8/200) of nuclei scored. DAPI signal has been pseudocolored white.

Effects of telomerase inhibition on hPOT1 dependent telomere lengthening. A. Inhibition of telomerase. BJ cells expressing a control hairpin (−) or a hairpin targeting hPOT1 (+) and either a second control hairpin (CONsh), one of two hairpins targeting hTERT (sh #1 or #2) or a dominant negative hTERT cDNA (DN) were analyzed by RT-PCR on RNA from cells synchronized in S-phase by serum starvation and release (COnsh, sh #1 and #2) or western blotting (CONsh and DN). B. hPOT1 mRNA levels. Relative hPOT1 expression was determined by quantitative RT-PCR on the cell lines described in panel A as well as HFF cells lines expressing a control hairpin (−) or a hairpin targeting hPOT1 (+) as well as CONsh or hTERTsh #1 or #2. C. TRF Southern of cells characterized in panels A and B. The mean TRF length at 14 population doublings after infection for each sample is indicated beneath the corresponding lane. Bar graphs show the change in telomere length after suppression of hPOT1 in the cell lines indicated. DNA size markers in kb are shown. D. Quantitative telomere FISH. Results of 12 metaphases for the BJ cell lines characterized in panels A–C are shown. The fluorescence intensity of telomeres from each sample was assigned into a bin the intensity of which is indicated on the X-axis, and the number of telomeres contained within that bin indicated on the Y-axis. The percent increase in mean fluorescence intensity for the hPOT1sh #1 versus CONsh for each pair of cell lines is indicated. Fluorescence values are arbitrary and cannot be compared between graphs.

Rescue of telomere length changes induced by hPOT1 suppression. A. Over-expression of hPOT1. hPOT1 mRNA levels in BJ or BJ hTERT cells expressing a control hairpin (CONsh) or an hPOT1 hairpin (hPOT1sh #1) in cells expressing the hPOT1sh #1 resistant version of hPOT1 (hPOT1R). B. TRF Southern blotting demonstrating rescue of hPOT1 suppression. The mean TRF length for each sample taken 10 PD after infection is indicated beneath the corresponding lane. p values (two-tailed t test) are shown for the indicated pair-wise comparisons from 3 (BJ) or 4 (BJ hTERT) replicate experiments. DNA size markers in kb shown on the left are for both gels. C. Quantitative telomere FISH demonstrating rescue of hPOT1 suppression. Results are shown for 12 metaphases for the BJ (left) and BJ hTERT (right) cell lines characterized in panels A and B. The fluorescence intensity of telomeres from each sample was assigned into a bin the intensity of which is indicated on the X-axis, and the number of telomeres contained within that bin indicated on the Y-axis. The percent increase in mean fluorescence intensity for the hPOT1sh #1 versus CONsh cell line is indicated. Fluorescence values are arbitrary and cannot be compared between graphs.

Long-term effects of hPOT1 suppression on telomere length and replicative senescence. A. TRF Southern of BJ cells over a span of 34 PD. BJ cells were infected with a control hairpin (−) or an hPOT1 hairpin (+) and DNA collected at the indicated mean population doubling after infection. BJ cells used in this experiment are of a lower initial starting PD from those in Figs. 1 & 2. The mean TRF length for each sample is indicated beneath the corresponding lane. DNA size markers in kb are shown on the left. B. TRF Southern of HFF cells over a span of 30 PD. HFF cells were infected with a control hairpin (c) or one of two hPOT1-specific hairpins (#1 or #2) and DNA collected at the indicated mean population doubling after infection. The mean TRF length for each sample is indicated beneath the corresponding lane. DNA size markers in kb are shown on the left. C. Long-term proliferation of BJ hTERT cells after hPOT1 suppression. BJ hTERT cells were infected with a control hairpin (CONsh) or one of two hPOT1 hairpins (hPOT1 #1 or #2). Cumulative PD measurements represent an average of three separate cultures. D. Long-term proliferation of BJ cells after hPOT1 suppression. BJ cells were infected with CONsh or hPOT1 #1 or #2. Cumulative PD measurements represent an average of three separate cultures and the p value (two-tailed t test) indicates the degree of statistical significance between the pair-wise comparison of CONsh and either hPOT1sh #1 or #2 (p ≤ 0.03 for both comparisons). E. SA β-gal staining. Values represent the percentage of positively staining cells of at least 100 total cells scored in triplicate of cells from d 69 of the proliferation experiment shown in panel D.