Telomerase elongates tiTELs independently of their transcription induction.

<p>(<b>A</b>) Cl12 and cl17 cells were treated with BIBR1532 for 60 population doublings and released into normal medium in presence or absence of DOX. STELA analysis of tiTELs was performed 0, 3, 6, 9, 15, 20 and 27 days (d) after release. Marker molecular weights are on the left in kilobases. (<b>B</b>) Whisker-boxplots represent STELA quantifications for each sample. Telomere elongation rates are expressed in base pairs per day (bp/d). All P-values between induced and uninduced samples were >0.1 and therefore are not indicated. For each condition, 77 to 400 telomeres were analyzed.</p

TERRA steady-state levels are not affected by telomere shortening in HeLa cells.

<p>(<b>A–D</b>) HeLa cells were treated with the telomerase inhibitor BIBR1532 (+BIBR) for 19 weeks or left untreated (−BIBR). Telomere length, TERRA promoter methylation and total or chromosome specific TERRA steady-state levels were analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035714#pone-0035714-g001" target="_blank">Figure 1</a>. Total RNA was used for all experiments. For northern blot analysis of total TERRA (<b>C</b>) beta-actin (ACT) was used as a normalization control. Molecular weights are on the left in kilobases.</p

TERRA steady-state levels are not affected by telomere elongation in HeLa cells.

<p>(<b>A</b>) TRF analysis of HeLa cells infected with empty vector (ev), hTERT or hTERT-HA retroviruses. DNA was digested with <i>Rsa</i>I and <i>Hinf</i>I restriction enzymes and hybridized with telomeric probes. (<b>B</b>) The same DNA as in <b>A</b> was digested with <i>Hpa</i>II (methylation sensitive) or <i>Msp</i>I (methylation insensitive) restriction nucleases and hybridized with a probe detecting the 29–37 bp repeats of TERRA promoters. (<b>C</b>) Nuclear RNA was hybridized using telomeric probes to detect total TERRA and successively with 18S rRNA probes to control for loading. Numbers at the bottom are the ratios between TERRA and 18S signal expressed as fold increase over ev-infected samples. Molecular weights are on the left in kilobases. (<b>D</b>) qRT-PCR analysis of the steady-state levels of TERRA transcripts originating from 10q, 15q and Xp/Yp chromosome ends. Bars are averages from three independent experiments expressed as fold increase over ev-infected samples. Error bars and numbers are standard deviations and P-values, respectively. (<b>E</b>) Western blot analysis of infected cells using anti-hTERT (to detect all hTERT molecules), anti-HA (to detect hTERT-HA) and anti-PCNA (loading control) antibodies.</p

Efficient telomerase-mediated elongation of telomeres in DKO cells.

<p>(<b>A</b>) TRF analysis of par and DKO cells infected with empty vector (ev) or hTERT retroviruses. Genomic DNA was collected 2, 5 and 9 days (d) after infection, digested with <i>Rsa</i>I and transferred to a nylon membrane. The same membrane was first hybridized with a probe detecting 10q TRFs and successively with a probe detecting total telomeres. (<b>B</b>) STELA of 15q telomere length using the same DNA as in <b>A</b>. Marker molecular weights are on the left in kilobases. (<b>C</b>) Box plot representation of 15q telomere lengths. Average telomere lengths in kilobases and the number of total telomeres analyzed (n) are indicated for each sample. (<b>D</b>) 15q telomere elongation rates expressed as average telomere length at different population doublings. Note that for par cells, only three time points were used because no statistically significant difference in average telomere length was measured between day 5 and day 9.</p

Generation and characterization of transcriptionally inducible telomeres (tiTELs).

<p>(<b>A</b>) Scheme of the tiTEL seeding vector. iCMV: inducible CMV promoter; SBF and SBR: oligonucleotides used in RT-PCR experiments; SBP: probe used in TRF, STELA and northern blot experiments. (<b>B</b>) tiTEL TRF analysis of genomic DNA prepared from clone 12 (cl12), clone 17 (cl17) and parental (par) cells infected with hTERT-expressing retroviruses or ev control retroviruses. DNA was hybridized using SBP probes. Marker molecular weights are on the left in kilobases. (<b>C</b>) Partial metaphases from cl12 and cl17 hybridized <i>in situ</i> to detect tiTELs (arrowheads). (<b>D</b>) Northern blot analysis of total RNA from cl12 and cl17 treated for 24 h with combinations of doxycycline (DOX) and trichostatin A (TSA) or left untreated. SBP probes were used to detect tiTERRA. The same membranes were stripped and re-probed to detect beta-actin transcripts (ACT) to control for loading. (<b>E</b>) qRT-PCR analysis of tiTERRA steady-levels in the indicated cell lines treated with different combinations of DOX and TSA or left untreated. For each cell line, values are expressed as fold increase over untreated samples. Bars and error bars are averages and standard deviations from 3 to 7 experiments. P values for relevant samples are indicated by numbers or by the asterisks (*: P<0.01; **: P<0.001).</p

Cells deficient for DNMT1 and 3b display normal telomerase activity.

<p>(<b>A</b>) Western blot analysis of hTERT-expression in HCT116 parental (par) or DNMT1 and 3b double KO (DKO) infected cells. Numbers at the bottom are the ratios between hTERT and PCNA (loading control) signals, expressed as fold increase over hTERT-infected parental cells. (<b>B</b>) qRT-PCR analysis of the steady-state levels of TERRA transcripts originating from 10q and 15q chromosome ends expressed as fold increase over ev-infected par cells. Bars and error bars are averages and standard deviations from three independent experiments. (<b>C</b>) Analysis of TRAP amplification products from the indicated cells lines. Three different amounts of total proteins were used for each cell line and to control for specificity one sample was pre-treated with RNase A. Control primers amplifying an internal control (IC) were included in all reactions. (<b>D</b>) Quantification of telomerase activity in the indicated samples using qRT-PCR-based TRAP assays. Bars and error bars are averages and standard deviations from three independent experiments, after normalization through ev-infected parental cell samples. Numbers indicate P-values (*: P<0.01). (<b>E</b>) qRT-PCR-based quantification of 10q and 15q TERRA transcripts in TRAP extracts (ext) or in cell pellets left after extraction (plt). Relative TERRA amounts are expressed as fractions of total TERRA molecules from pellet plus extract. Bars and error bars are averages and standard deviations from three independent experiments.</p