Fig 1.
Induction of telomere damage upon etoposide treatment.
A. Immunoblot of γ-H2AX in HeLa cells treated with etoposide for the indicated times; β-actin was used as a loading control. B. ChIP results for telomeric and centromeric DNA with the anti-γ-H2AX antibody in HeLa cells treated with etoposide for the indicated times. DNA slot blots were hybridized with telomere- or centromere-specific probes. Antibodies specific for γ-H2AX and control IgG were used for the ChIP assays. C. Quantification of three independent ChIP assays represented in B (mean ± standard deviation, SD). Mann-Whitney U-test was used to compare differences in DNA levels between each time point and 0 h; *P < 0.05.
Fig 2.
Increased levels of TERRAs in etoposide-treated HeLa cells.
A. RT–qPCR analysis of TERRAs. HeLa cells were treated with 100 μM of etoposide or vehicle alone (DMSO) for the indicated times. TERRAs were measured using RT–qPCR with 11 pairs of subtelomere-specific primers as indicated. The levels of TERRAs were normalized to 18S rRNA and compared with 0 h. Error bars are SD derived from at least three independent assays. Mann-Whitney U-test was used to compare TERRA levels between each time point and 0 h; an asterisk (*) on the bars denotes P < 0.05. One-way ANOVA was used for comparing TERRA levels at various incubation times for each representative subtelomere; **P < 0.05, and ns P ≥ 0.05. B. Northern blot analysis of TERRAs. Total RNAs were isolated from HeLa cells treated with etoposide for the indicated times. The DIG-labeled probes used in hybridization are indicated. The membrane was reprobed after stripping off the previous probes. Size markers, 28S and 18S rRNAs, are indicated. The d(CCCTAA)4-DIG-specific diffuse bands, indicated by a bracket, were quantified, normalized to 18S bands, and compared with 0 h. C. Quantification of northern blot analyses in B (mean ± SD; n = 4). One-way ANOVA was used for comparing TERRA levels according to different incubation times (P = 0.004). Mann-Whitney U-test was used to compare differences in TERRA levels between each time point and 0 h; *P < 0.05.
Fig 3.
Telomere length and TRF2-mediated telomere integrity were maintained in etoposide-treated HeLa cells.
A. Southern blot analysis of telomeres. Terminal restriction fragments were detected in HeLa cells treated with etoposide for the indicated times. The blot was hybridized with a DIG-labeled d(TTAGGG)4 probe (left panel), stripped, and then rehybridized with a DIG-labeled d(CACCAC)4 probe (right panel). Size markers are indicated on the left. B. Telomere length in HeLa cells treated with etoposide. The telomere lengths were determined as described previously [27], and the mean telomere length was obtained from two independent experiments (mean ± SD). C. ChIP assays in HeLa treated with etoposide for the indicated hours. DNA precipitates were slot-blotted and hybridized with telomeric and alphoid repeat-containing probes. Antibodies specific for TRF2 and control IgG were used for the ChIP assays. D. Quantification of ChIP assays represented in C (mean ± SD; n = 3).
Fig 4.
ChIP–qPCR to assay RNAPII binding at subtelomeres after exposure to etoposide.
HeLa cells incubated with etoposide at 100 μM for the indicated times were subjected to ChIP assays using an anti-phosphorylated RNAPII antibody (pS2) and IgG as a control. Associated DNA fragments were PCR-amplified for p21, GAPDH, and subtelomeres as indicated; p21-11 and p21-12 indicate the p21 gene at 0.2 and 2.1 kb from the transcription start site, respectively. Results are presented as percentages of input DNA. Bars represent the SDs of at least three replicates. Student’s t-test was used to compare DNA levels between RNAPII-ChIP and IgG-ChIP; **P < 0.05 and ns indicates P ≥ 0.05. Mann-Whitney U-test was used to compare DNA levels between each h and 0 h; *P < 0.05. B. RT–qPCR for p21 and GAPDH in HeLa cells treated with DMSO (vehicle alone) or etoposide at 100 μM for the indicated times. p21 and GAPDH levels were normalized to 18S rRNA and expressed as fold changes relative to 0 h. Mann-Whitney U-test was used to compare DNA levels between each h and 0 h; *P < 0.05.
Fig 5.
Stability of TERRAs in etoposide-treated cells.
HeLa cells incubated with DMSO (vehicle alone) or etoposide at 100 μM for 24 h were treated with actinomycin D at 5 μg/mL for the indicated times. TERRAs were measured using RT–qPCR with primers as indicated, normalized against 18S rRNA, and compared with 0 h. Error bars are SDs derived from at least five independent experiments. Student’s t-tests were used to compare differences in TERRA levels between etoposide- and DMSO-treated cells; *P < 0.05.