Nuclear TERT localization correlates with high DNA damage levels after treatment with H₂O₂

<p><b>while mitochondrial telomerase prevents it. A–C:</b> Representative images of TERT localization (green), and γH2A.X staining (red). Blue: DAPI nuclear counterstain <b>A:</b> HeLa <b>B:</b> MCF7 <b>C:</b> MRC-5/hTERT cells. Cells were treated for 3 h with 400 µM H₂O₂. TERT localization was determined as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052989#pone-0052989-g001" target="_blank">Figure 1B</a> and grouped into 3 categories: nuclear TERT (N) TERT (C) and intermediary TERT (I) localization. Examples for the 3 different localizations are indicated with arrows. <b>D:</b> Correlation between subcellular TERT localization and nuclear DNA damage levels (number of γH2A.X foci). Cytoplasmic TERT localization correlates with low nuclear DNA damage in all 3 cell lines while nuclear TERT localization results in high nuclear damage after 3 h of treatment with 400 µM H₂O₂. Intermediary TERT localization results in intermediate DNA damage levels. Black bars: HeLa, red bars: MCF7, green bars: MRC-5/hTERT. Bars are mean ± SE from at least 40–100 cells per cell line in repeated experiments. * P<0.05.</p

Mitochondrially localized TERT protects against mitochondrial ROS generation after H₂O₂ treatment and irradiation in 4 different cell lines. A:

<p>Upper panel: Representative images of ROS staining (red, mitosox) and TERT localization (myc-tag, green) after organelle specific TERT transfection and 100 µM H₂O₂ treatment for 3 h in HeLa cells. Upper row: mito- TERT, lower row: nuclear TERT. Arrows indicate transfected cells. Lower panel: Quantification of ROS levels measured as percentage of mitosox positive area from whole cytoplasm using ImageJ in transfected and un-transfected cells. <b>B:</b> MCF7 cells, panels as described for A. <b>C:</b> Quantification of ROS in U87 cells after 3 h of 100 µM H₂O₂ treatment. <b>D–F:</b> Quantification of ROS levels after X-irradiation. <b>D:</b> MCF7 after 20 Gy X-irradiation. <b>E:</b> U87 after 20 Gy X-irradiation <b>F:</b> MRC-5/SV40 after 10 Gy X-irradiation. Bars represent mean ± SE from 3 independent experiments. * P<0.05.</p

Mitochondrial TERT protects from apoptosis induction after H₂O₂ treatment and X-irradiation compared to cells transfected with nuclear TERT.

<p>Representative images of activated caspase 3 (shown in red) in <b>A</b>: Hela, <b>B</b>: MRC/SV40, <b>C</b>: U87 cells transfected with mito TERT and nuclear TERT (myc-tag, shown in green) after 400 µM H₂O₂ treatment for 3 h or irradiation with 20 Gy. <b>D</b>: Quantification of the percentage of apoptotic cells of the 3 cell lines after H₂O₂ treatment, E: Quantification of the percentage of apoptotic cells of the 3 cell lines after X-irradiation. Bars present mean and standard error from around 45 transfected cells per condition and cell line. * p<0.05.</p

TERT shuttles from nucleus to mitochondria upon H₂O₂ treatment in cancer cells. A:

<p>Example rendered 3D volume projections of deconvolved confocal images from HeLa and MCF7 cells untreated (control, left panel) or treated with 400 µM H₂O₂ for 3 h (right panel). Green represents mitotracker green fluorescence, red anti-TERT immuno-fluorescence and blue nuclear DNA (DAPI). Marked colocalization between mitotracker green and TERT is displayed by red-green mixing being displayed as yellow. <b>B–D:</b> TERT localization kinetics in 3 cell line populations after treatment with 400 µM H₂O₂ over 5 days. <b>B:</b> HeLa <b>C:</b> MCF7 <b>D:</b> MRC-5/hTERT. Black bars: nuclear TERT, red bars: cytoplasmic TERT. Bars are means ± SE from at least 30 cells per time point and cell line from 3 independent experiments.</p

Mitochondrially located TERT reduces nuclear DNA damage after

<p>H₂O₂<b>treatment in comparison to nuclear TERT localization in 4 different cell lines. A</b>: Organelle specific TERT vectors transfected into HeLa cells. Upper panel: representative images of cells transfected with mitochondrial and nuclear TERT shooter vectors with and without treatment with 200 µM H₂O₂ for 3 hours. TERT staining (using myc-tag) fused to TERT protein (green) and γH2A.X staining (red) for DNA damage foci. Arrows show transfected cells. Lower panel: Quantification of cells with high levels of DNA damage foci for transfected and un-transfected cells with and without H₂O₂ treatment. Bars are mean ± SE from 3 independent experiments, *P<0.05. <b>B</b>: Organelle specific TERT vectors transfected into MCF7 cells. Panels as described for A. <b>C–F</b>: Quantification of cells with high levels of DNA damage foci for transfected and un-transfected cells with and without x-irradiation. <b>C</b>: MCF7 after 20 Gy X- irradiation. <b>D:</b> U87 after 20 Gy X-irradiation. <b>E</b>: MRC-5/SV40 after 10 Gy X-irradiation. Bars are mean ± SE from 3 independent experiments. * P<0.05.</p

Quantification and significances of z-stacks for determination of correlation coefficient for co-localization of hTERT to mitochondria in three cell lines.

<p>Mean correlation coefficients were determined from between 8–10 cells per cell line/treatment. Pearson correlation coefficients were determined per cell for deconvolved, 3D rendered images, subtracting any background for each channel, using Huygens Colocalization analyzer plugin (Huygens, SVI, Netherlands). All datasets passed Normality tests and P values are from Student's T tests comparing untreated and treated cells per cell line.</p