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

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 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

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

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

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

AR expression and activity within the prostate epithelial hierarchy of differentiation.

<p>A) Representative images of expression of CD133 and AR counterstained with DRAQ5™ within prostate EpCAM^+VE α₂β₁^HI CD133^+VE (Left panel), α₂β₁^HI CD133^–VE (central panel) and α₂β₁^LOW CD133^–VE cells (right panel). <b>B)</b> Expression of the AR regulated genes PSA, KLK2 and TMPRSS2 normalised to GAPDH (n = 10) (p<0.001 comparing α₂β₁^HI and α₂β₁^LOW). Error bars represent standard error of the mean.</p

Expression of the AR within the prostate epithelial hierarchy of differentiation.

<p>Error bars represent standard error of the mean. <b>A)</b> Expression of AR transcript relative to GAPDH. Error bars represent standard error of the mean for n = 10. <b>B)</b> Upper dotplot representative of CD133 staining for progenitor α₂β₁^HI cells. Lower left dotplot representative of AR expression in CD133^–VE gated α₂β₁^HI cells. Lower right dotplot representative of AR expression in CD133^+VE gated α₂β₁^HI cells. Gates were set according to appropriate isotype controls. <b>C)</b> Mean percentage of cells expressing the AR in CD133^+VE and CD133^–VE α₂β₁^HI cells (n = 6). <b>D)</b> Representative histograms for fluorescence of α₂β₁^HI and α₂β₁^LOW isotype controls and AR detection. <b>E)</b> Mean fold change in median staining relative to isotype control for AR stained α₂β₁^HI cells and α₂β₁^LOW cells (n = 6).</p

Validation of AR detection with flow cytometry.

<p><b>A)</b> Percentage of cells staining above a no antibody control for either isotype antibody (hollow points) or PG-21 AR antibody (solid points) in LNCaP (circles) or PC3 (triangles) across a dilution series. <b>B)</b> Representative staining patterns for PC3 (upper dotplots) and LNCaP (lower dotplots) for either 1∶200 isotype antibody (left dotplots) or 1∶200 PG-21 AR antibody (right dotplots) of equivalent concentrations. Gates set according to isotype control. <b>C)</b> Left dotplot representative of staining of LNCaP with isotype control, right dotplots representative of AR staining in non-transfected LNCaP (upper), LNCaP transfected with scrambled siRNA (middle dotplot) and LNCaP transfected with AR siRNA (lower). Gates were set according to an appropriate isotype control. <b>D)</b> Percentage of cells staining positive for AR relative to an isotype control in non-transfected LNCaP, LNCaP transfected with scrambled siRNA and LNCaP transfected with AR siRNA. Error bars represent standard error of the mean for n = 3. <b>E)</b> Western blots of AR expression for non-transfected LNCaP, LNCaP transfected with scrambled siRNA and LNCaP transfected with AR siRNA are shown using a different AR antibody (G122-434, BD Pharmingen).</p

Strategy of enrichment for required cell types.

<p><b>A)</b> Schematic work flow for enrichment of epithelial cells for assessment of AR expression. <b>B)</b> Purity of selection by expression of the lineage specific markers CD24 (epithelial), CD146 (endothelial) and CD45 (haematopoietic) normalised to GAPDH following real-time PCR for unsorted prostate epithelia and EpCAM/HEA sorted epithelia, error bars represent standard error of the mean for n = 3. <b>C)</b> CD133/1 Sorted samples were assessed for purity by co-expression of the CD133/2 epitope, confirming that these two epitopes are co-expressed in the prostate and that our CD133 selection efficiently enriches for CD133^+VE cells: Upper dotplot representative of CD133/2 staining for unsorted α₂β₁^HI epithelial cells; lower left dotplot representative of CD133/2 staining for α₂β₁^HI CD133/1^–VE cells; lower right dotplot representative of CD133/2 staining for α₂β₁^HI CD133/1^+VE cells. Gates are set according to appropriate isotype controls. <b>D)</b> Confirmation of CD133 enrichment with real-time PCR. CD133 expression is shown normalised to GAPDH, error bars represent standard error of the mean n = 10.</p