Journal: Oncotarget

Article Title: Bortezomib-mediated down-regulation of telomerase and disruption of telomere homeostasis contributes to apoptosis of malignant cells.

doi: 10.18632/oncotarget.5752

Figure Lengend Snippet: Figure 2: Widespread dysregulation of shelterin protein expression in bortezomib-treated HEL and BGC-823 cells. A. and B. mRNA levels of shelterin factors TRF1, TRF2, TPP1, POT1, RAP1 and TIN2 in bortezomib-treated cells. Cells were treated with bortezomib for 24 hours and qPCR was used for quantitative assays. The levels of each target mRNA in bortezomib-treated cells were expressed as percentages of those in untreated cells. (A) HEL cells and (B) BGC-823 cells. C. and D. Immunoblotting assessment of TRF1, TRF2 and POT1 protein expression in bortezomib-treated cells. Same sets of cells above were analyzed for TRF1, TRF2 and POT1 protein levels and shown was representative of three independent experiments. (C) HEL cells and (D) BGC-823 cells. * and **: P < 0.05 and 0.01, respectively. BTZ, bortezomib.

Article Snippet: The membranes were probed with the specific antibodies against TRF1 (SigmaAldrich, St. Louis, MO, USA, T1948), TFR2 (Novus, Littleton, CO, USA, #NB110–57130), POT1 (Novus, NB500–176), Caspase3 (Cell Signaling Technology, Danvers, MA, USA, #9665), or Bcl-2 (ProteinTech, Wuhan, China, #12789–1-AP) followed by anti-mouse or rabbit horseradish peroxidase-conjugated IgG and developed with the enhanced chemiluminescent method (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific).

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: Bortezomib-mediated down-regulation of telomerase and disruption of telomere homeostasis contributes to apoptosis of malignant cells.

doi: 10.18632/oncotarget.5752

Figure Lengend Snippet: Figure 4: Attenuation of bortezomib-induced shelterin protein dysregulation by hTERT over-expression. A. and B. Cells expressing ectopic hTERT were treated with bortezomib for 24 hours and mRNA levels of shelterin proteins then analyzed using qPCR. The levels of each target mRNA in bortezomib-treated cells were expressed as percentages of those in untreated cells. (A) HEL-hTERT cells and (B) BGC-823-hTERT cells. C. and D. Immunoblotting assessment of TRF1, TRF2 and POT1 protein expression in bortezomib-treated cells. Same sets of cells above were analyzed for TRF1, TRF2 and POT1 protein levels and shown was representative of three independent experiments. (C) HEL-hTERT cells and (D) BGC-823-hTERT cells.* and **: P < 0.05 and 0.01, respectively. BTZ, bortezomib.

Article Snippet: The membranes were probed with the specific antibodies against TRF1 (SigmaAldrich, St. Louis, MO, USA, T1948), TFR2 (Novus, Littleton, CO, USA, #NB110–57130), POT1 (Novus, NB500–176), Caspase3 (Cell Signaling Technology, Danvers, MA, USA, #9665), or Bcl-2 (ProteinTech, Wuhan, China, #12789–1-AP) followed by anti-mouse or rabbit horseradish peroxidase-conjugated IgG and developed with the enhanced chemiluminescent method (SuperSignal West Pico Chemiluminescent Substrate, Thermo Scientific).

Techniques: Over Expression, Expressing, Western Blot

Journal: Nature Communications

Article Title: The telomere binding protein Pot1 maintains haematopoietic stem cell activity with age

doi: 10.1038/s41467-017-00935-4

Figure Lengend Snippet: Expression of Pot1a in HSPCs. a Expression of Pot1a in: Lineage + (Lin + ) cells; Lin − Kit + Sca-1 − (LKS − ) cells; LSKCD41 + CD48 + CD150 − multipotent progenitor (MPP) cells; LSKCD41 + CD48 + CD150 + cells (ST-HSCs); LSKCD41 − CD48 − CD150 + cells (LT-HSCs) isolated from 8 week-old mice. Data are expressed as the mean ± SD ( n = 4, * p < 0.01, ** p < 0.05 by Tukey’s test). Representative data from two independent experiments are shown. b Immunocytochemical staining of POT1 (red) in HSPCs. Nucleus is stained by TOTO3 (blue). Scale bar, 2 μm. c Relative area of POT1 immunofluorescent dots in HSPCs. Fluorescence images were analyzed with ImageJ. Data are expressed as the mean ± SD ( n = 64: MPP, n = 67: ST-HSCs, n = 54: LT-HSCs, * p < 0.01 by Tukey’s test). Representative data from two independent experiments are shown. d Immunoblot analysis of POT1 in Lin + , Lin – , LSKCD150 – , and LSKCD150 + cells (left panel). Densitometry analysis of the immunoblot is shown in the right panel . Representative data from two independent experiments are shown. e Expression of Pot1a in 8, 60, and 90 week-old LT-HSCs. Data are expressed as the mean ± SD ( n = 4, * p < 0.01 by Tukey’s test). Representative data from two independent experiments are shown. f Immunocytochemical staining of POT1 (red) in LT-HSCs isolated from 8 and 90 week-old mice. Scale bar , 2 μm. g Relative area of POT1 immunofluorescent dots in LT-HSCs isolated from 8 and 90 week-old mice. Data are expressed as the mean ± SD ( n = 146: 8-week-old, n = 180: 90-week-old, * p < 0.01 by t -test)

Article Snippet: For the construction of the retrovirus vector-expressing mouse Pot1a, full-length cDNA of mouse Pot1a (OriGene Technologies, Inc.) was ligated into pMY-IRES-GFP (provided by Dr. Kitamura; University of Tokyo, Institute of Medical Science) or pMY-IRES-monomeric Kusabira-Orange1 (mKO1).

Techniques: Expressing, Isolation, Staining, Fluorescence, Western Blot

Journal: Nature Communications

Article Title: The telomere binding protein Pot1 maintains haematopoietic stem cell activity with age

doi: 10.1038/s41467-017-00935-4

Figure Lengend Snippet: Overexpression of Pot1a enhances HSC proliferation activity. a – c Control GFP or Pot1a-transduced LSK cells isolated from 8 week-old mice were cultured for 2 weeks and their ability to form colonies was analyzed. a Representative colonies from control GFP ( left ) and Pot1a-transduced LSK cells ( right ) after 2 weeks in vitro culture. Scale bar , 0.5 mm. b Number of CFU-Cs. c Number of HPP-CFCs. Data are expressed as the mean ± SD ( n = 3, * p < 0.01 by t -test). d , e Control GFP or Pot1a-transduced LSK cells were clone-sorted and cultured for 2 weeks in SF-O3 medium supplemented with SCF (20 ng ml –1 ), TPO (50 ng ml –1 ), IL-3 (20 ng ml –1 ) and EPO (1 U ml –1 ). d Development of colonies from single cells after 2 weeks in vitro culture. e Number of colonies derived from single cells after 1–3 weeks in vitro culture. Each colony was classified into three categories by their size (<1 mm, ≥1 mm, ≥2 mm). Data are expressed as the mean ± SD ( n = 3, * p < 0.01, ** p < 0.05 by Tukey’s test). f Control GFP or Pot1a-transduced 8 week-old LSK cells were cultured for 7–14 days. Subsequently, 5 × 10 3 GFP + LSK cells were transplanted into lethally irradiated mice. The number of recipient mice that showed long-term reconstitution of donor cells (>1% of GFP + Ly5.1 + cells in PB) is shown

Article Snippet: For the construction of the retrovirus vector-expressing mouse Pot1a, full-length cDNA of mouse Pot1a (OriGene Technologies, Inc.) was ligated into pMY-IRES-GFP (provided by Dr. Kitamura; University of Tokyo, Institute of Medical Science) or pMY-IRES-monomeric Kusabira-Orange1 (mKO1).

Techniques: Over Expression, Activity Assay, Control, Isolation, Cell Culture, In Vitro, Derivative Assay, Irradiation

Journal: Nature Communications

Article Title: The telomere binding protein Pot1 maintains haematopoietic stem cell activity with age

doi: 10.1038/s41467-017-00935-4

Figure Lengend Snippet: Overexpression of Pot1a maintains HSC LTR activity. a , b Results of primary BMT. a Percentage of donor-derived (Ly5.1 + GFP + ) cells in recipient mice PB, BM LSK and LT-HSCs 4 months after BMT. b Peripheral blood analysis of B, T, and myeloid cell lineages 4 months post BMT. Data are expressed as the mean ± SD ( n = 5 group –1 , ** p < 0.05 by t -test). Representative data from two independent experiments are shown. c , d Results of secondary BMT. c The percentage of donor-derived (Ly5.1 + GFP + ) cells in recipient mice PB, BM LSK and LT-HSCs 4 months post secondary BMT. d Peripheral blood analysis of B, T, and myeloid cell lineages 4 months post secondary BMT. Data are expressed as the mean ± SD ( n = 5, * p < 0.01 by t -test). Representative data from two independent experiments are shown

Article Snippet: For the construction of the retrovirus vector-expressing mouse Pot1a, full-length cDNA of mouse Pot1a (OriGene Technologies, Inc.) was ligated into pMY-IRES-GFP (provided by Dr. Kitamura; University of Tokyo, Institute of Medical Science) or pMY-IRES-monomeric Kusabira-Orange1 (mKO1).

Techniques: Over Expression, Activity Assay, Derivative Assay

Journal: Nature Communications

Article Title: The telomere binding protein Pot1 maintains haematopoietic stem cell activity with age

doi: 10.1038/s41467-017-00935-4

Figure Lengend Snippet: Pot1a prevents DDR in HSCs. a , b Telomeric DDR in 8 week-old LSK cells upon Pot1a knockdown. a Immunocytochemical staining of TRF1 ( green ) and 53BP1 ( red ). Foci co-stained with TRF1 and 53BP1 were identified as TIFs. Nuclei were stained with TOTO3 ( blue ). Scale bar , 2 μm. b Frequency of TIFs per cell after 1 week of culture. Data are expressed as the mean ± SD ( n = 100, * p < 0.01 by t -test). Representative data from three independent experiments are shown. c – f Telomeric DDR in donor-derived control-GFP or Pot1a overexpressing 8 week-old LSK cells 4 months post BMT. c Immunocytochemical staining of TRF1 ( green ) and 53BP1 ( red ). Nuclei were stained with TOTO3 ( blue ). Scale bar , 2 μm. d Frequency of TIFs per cell. Data are expressed as the mean ± SD ( n = 120, * p < 0.01 by t -test). Representative data from 3 independent experiments are shown. e Immunocytochemical staining of TRF1 ( green ) and RPA32 ( red ). f Immunocytochemical staining of TRF1 ( green ) and pChk1 ( red ). Scale bar , 2 μm. g Flow cytometric analysis of Chk1 and pChk1 in donor-derived GFP + LSKCD48 – CD150 + cells after 5 months of 2nd BMT. Mean fluorescence intensity of Chk1 and pChk1 ( left panels ). Data are expressed as the mean ± SD ( n = 3, * p < 0.01 by t -test). Representative FACS profiles of Chk1 and pChk1 in donor-derived GFP + LSKCD48 – CD150 + cells ( right panels )

Article Snippet: For the construction of the retrovirus vector-expressing mouse Pot1a, full-length cDNA of mouse Pot1a (OriGene Technologies, Inc.) was ligated into pMY-IRES-GFP (provided by Dr. Kitamura; University of Tokyo, Institute of Medical Science) or pMY-IRES-monomeric Kusabira-Orange1 (mKO1).

Techniques: Knockdown, Staining, Derivative Assay, Control, Fluorescence

Journal: Nature Communications

Article Title: The telomere binding protein Pot1 maintains haematopoietic stem cell activity with age

doi: 10.1038/s41467-017-00935-4

Figure Lengend Snippet: Effect of exogenous Pot1a on ROS production in LT-HSCs in culture. a Schematic of the microarray analysis. b GSEA plots demonstrating enrichment levels of indicated gene sets in control control-GFP versus Pot1a-transduced LT-HSCs. NES, NOM p value, and FDR are indicated. c Schematic of the measurement of ROS in LT-HSCs. d Intracellular ROS levels in control-GFP ( blue ) and Pot1a-transduced ( red ) LT-HSCs. Representative FACS profiles of CellROX in GFP + LT-HSCs are shown. e Mean fluorescence level. Data are expressed as the mean ± SD ( n = 5, * p < 0.01 by t -test). Representative data from two independent experiments are shown. f Relative mean fluorescence level of MitoSOX in the GFP + LT-HSCs on days 4, 7, and 11 of culture. Mean fluorescence level of MitoSOX in control-GFP-transduced LT-HSCs was set to 1.0. Data are expressed as the mean ± SD ( n = 6: day 7, n = 12: day 7, n = 3: day 11, * p < 0.01 by Tukey’s test). g Schematic of the measurement of ROS in LT-HSCs upon overexpression or knockdown of Pot1a. h Expression of Mtor and Rptor in Pot1a-overexpressing LT-HSCs. Data are expressed as the mean ± SD ( n = 4, * p < 0.01 by t -test). i Expression of Mtor and Rptor in Pot1a knockdown LT-HSCs. Data are expressed as the mean ± SD ( n = 4, * p < 0.01 by t -test)

Article Snippet: For the construction of the retrovirus vector-expressing mouse Pot1a, full-length cDNA of mouse Pot1a (OriGene Technologies, Inc.) was ligated into pMY-IRES-GFP (provided by Dr. Kitamura; University of Tokyo, Institute of Medical Science) or pMY-IRES-monomeric Kusabira-Orange1 (mKO1).

Techniques: Microarray, Control, Fluorescence, Over Expression, Knockdown, Expressing

Journal: Nature Communications

Article Title: The telomere binding protein Pot1 maintains haematopoietic stem cell activity with age

doi: 10.1038/s41467-017-00935-4

Figure Lengend Snippet: Treatment with exogenous POT1 protein protects LT-HSCs in culture. a Effect of MTM-POT1a on colony formation of HSCs. LT-HSCs (8 week-old) were cultured with MTM-POT1a for 2 weeks. After culture, Lin − cells were isolated and re-cultured in MethoCult™ GF M3434 medium (200 cells per dish). Data are expressed as the mean ± SD ( n = 3, * p < 0.01 by Tukey’s test). Representative data from two independent experiments are shown. b , c LT-HSCs (8 week-old) were cultured with MTM-POT1a or control MTM protein. b Number of total cells and LT-HSCs on day 4, 7, and 10 of culture. Data are expressed as the mean ± SD ( n = 6, * p < 0.01 by t -test). c Percentage of Annexin V + PI + apoptotic cells in LT-HSCs on day 4, 7, and 10 of culture. Data are expressed as the mean ± SD ( n = 6, * p < 0.01 by t -test). d LT-HSCs (8 week-old) were transduced with control shRNA or shPot1a-1. After 2 days of shRNA transduction, GFP + LT-HSCs were re-sorted and cultured with MTM-POT1a or control MTM protein. After 1 week of culture, Annexin V assay was performed. Percentage of Annexin V + PI + apoptotic cells in GFP + LT-HSC fraction is shown. ( n = 3, ** p < 0.05 by Tukey’s test). e Immunocytochemical staining of TRF ( green ), 53BP1 ( red ), and TOTO3 ( blue ) in 8 week-old LT-HSCs cultured for 3 weeks ( left ). Frequencies of TIFs after 3 weeks of culture ( right ). Data are expressed as the mean ± SD ( n = 80–100, * p < 0.01 by t -test). f Representative FACS profiles of 8 week-old LT-HSCs after 3 weeks of culture with control MTM protein or MTM-POT1a ( left ). Numbers of LSK cells and LT-HSCs after 3 week culture (starting from 2400 cells) are shown in right panels. Data are expressed as the mean ± SD ( n = 3, * p < 0.01 by t -test). Representative data from five independent experiments are shown

Article Snippet: For the construction of the retrovirus vector-expressing mouse Pot1a, full-length cDNA of mouse Pot1a (OriGene Technologies, Inc.) was ligated into pMY-IRES-GFP (provided by Dr. Kitamura; University of Tokyo, Institute of Medical Science) or pMY-IRES-monomeric Kusabira-Orange1 (mKO1).

Techniques: Cell Culture, Isolation, Control, Transduction, shRNA, Annexin V Assay, Staining

Journal: Nature Communications

Article Title: The telomere binding protein Pot1 maintains haematopoietic stem cell activity with age

doi: 10.1038/s41467-017-00935-4

Figure Lengend Snippet: MTM-POT1a maintains self-renewal activity of HSCs. a Results of primary BMT. Percentage of donor-derived (Ly5.1 + ) cells in PB and LT-HSC fractions 4 months after BMT. b Frequency of donor cells in T, B, and myeloid cell fractions in PB 4 months post BMT. Data are expressed as the mean ± SD ( n = 5 group –1 , * p < 0.01, ** p < 0.05 by t -test). Representative data from four independent experiments are shown. c Results of secondary BMT. Percentage of donor-derived (Ly5.1 + ) cells in PB and LT-HSC fractions 4 months post secondary BMT. d Frequency of donor cells in T, B, and myeloid cell fractions in PB 4 months post secondary BMT. Data are expressed as the mean ± SD ( n = 5 per group, * p < 0.01 by t -test). Representative data from four independent experiments are shown. e Effect of mutant forms of POT1a in the regulation of LTR activity of HSCs. Percentage of donor-derived (Ly5.1 + ) cells in PB 4 months after BMT is shown. Data are expressed as the mean ± SD ( n = 5 group –1 , * p < 0.01 by Tukey’s test). Representative data from two independent experiments are shown. f Effect of the overexpression of mutant forms of Pot1a on the maintenance of LTR activity of HSCs. Percentages of GFP + donor-derived cells in PB 5 months after BMT are shown. Data are expressed as the mean ± SD ( n = 5 per group, * p < 0.01 by Tukey’s test). Representative data from two independent experiments are shown. g Result of limiting dilution competitive BMT. A log-fraction plot of the limiting dilution model is shown. The slope of the line is the log-active cell fraction. The dotted lines give 95% confidence intervals ( n = 24: control 20 cells, n = 21: control 50 cells, n = 23: MTM-POT1a 20 cells, n = 23: MTM-POT1a 50 cells). Competitive repopulation units are also shown

Article Snippet: For the construction of the retrovirus vector-expressing mouse Pot1a, full-length cDNA of mouse Pot1a (OriGene Technologies, Inc.) was ligated into pMY-IRES-GFP (provided by Dr. Kitamura; University of Tokyo, Institute of Medical Science) or pMY-IRES-monomeric Kusabira-Orange1 (mKO1).

Techniques: Activity Assay, Derivative Assay, Mutagenesis, Over Expression, Control

Journal: Nature Communications

Article Title: The telomere binding protein Pot1 maintains haematopoietic stem cell activity with age

doi: 10.1038/s41467-017-00935-4

Figure Lengend Snippet: MTM-POT1a improves aged HSC function. a Schematic of the analysis of cell number and apoptotic fraction of 90 week-old LT-HSCs after culture with MTM-POT1a or control MTM protein. b Number of total cells and LT-HSCs on days 4, 7, and 10 of culture. Data are expressed as the mean ± SD ( n = 5: day 4, n = 6: day 7 and 10, ** p < 0.05 by t -test). c Percentage of Annexin V + PI + apoptotic cells in LT-HSC fraction on day 4, 7, and 10 of culture. Data are expressed as the mean ± SD ( n = 6–8). d , e Effect of MTM-POT1a on reconstitution after BMT of young and aged LT-HSCs. 8 and 90 week-old LT-HSCs were cultured with MTM-POT1a or control MTM protein for 10 days and transplanted into lethally irradiated mice. d Percentages of donor-derived (Ly5.1 + ) cells in PB and LT-HSCs 4 months after BMT are shown ( n = 5 per group, * p < 0.01, ** p < 0.05 by Tukey’s test). Representative data from two independent experiments are shown. e Percentage of T cells (CD3 + ), B cells (B220 + ), and myeloid (Mac-1 + /Gr-1 + ) cells in donor-derived cells in PB ( n = 5 group –1 , * p < 0.01, ** p < 0.05 by Tukey’s test). Representative data from 2 independent experiments are shown. f 60 week-old LT-HSCs were cultured with MTM-POT1a or control MTM protein for 3 days. After the culture, gene expression profiles were analyzed by microarray. GSEA plots demonstrating enrichment levels of indicated gene sets in control MTM protein treated cells versus MTM-POT1a treated cells. NES, NOM P value, and FDR are indicated

Article Snippet: For the construction of the retrovirus vector-expressing mouse Pot1a, full-length cDNA of mouse Pot1a (OriGene Technologies, Inc.) was ligated into pMY-IRES-GFP (provided by Dr. Kitamura; University of Tokyo, Institute of Medical Science) or pMY-IRES-monomeric Kusabira-Orange1 (mKO1).

Techniques: Control, Cell Culture, Irradiation, Derivative Assay, Gene Expression, Microarray

Journal: Oncology Letters

Article Title: Trastuzumab increases the sensitivity of HER2-amplified human gastric cancer cells to oxaliplatin and cisplatin by affecting the expression of telomere-associated proteins

doi: 10.3892/ol.2014.2793

Figure Lengend Snippet: Effect of trastuzumab administration on the expression of telomere-associated genes and proteins in NCI-N87 cells. (A) Reverse transcription-quantitative polymerase chain reaction was used to determine the levels of TPP1 , POT1 , TRF1 , TRF2 , TERF2IP and TINF2 mRNA expression in NCI-N87 cells treated with trastuzumab and cisplatin (alone or in combination). Error bars indicate the standard deviation from the mean. * P<0.05, vs. control cells. (B) Western blot analysis determined that the protein expression levels of TPP1, POT1 and TRF2 in NCI-N87 cells were significantly inhibited by various concentrations of trastuzumab alone and low-dose trastuzumab in combination with cisplatin. TPP1 (formerly known as TINT1, PTOP and PIP); POT1, protection of telomere 1; TRF2, telomeric repeat binding factor 2; Control, untreated NCI-N87 cells.

Article Snippet: Following determination of protein concentration using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Inc.), an aliquot of lysate containing 50 μg of each protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, blocked with blocking buffer (PBS Tween-20 containing 5% non-fat milk) for 2 h at room temperature and incubated overnight at 4°C with the following specific primary antibodies: monoclonal mouse anti-human TPP1 (ACD; cat. no. TA504406), monoclonal rabbit anti-human POT1 (cat. no. TA310771), polyclonal rabbit anti-human TRF1 (cat. no. TA322887), rabbit anti-human monoclonal TRF2 (cat. no. TA307200), rabbit anti-human polyclonal TIN2 (cat. no. TA315321), rabbit anti-human polyclonal TRF2IP (cat. no. TA324532 ) and rabbit anti-human polyclonal GAPDH (cat. no. TA308884) (1:10,000, all primary antibodies; OriGene Technologies, Inc., Rockville, MD, USA).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation, Control, Western Blot, Binding Assay

Journal: bioRxiv

Article Title: Intercellular telomere transfer extends T cell lifespan

doi: 10.1101/2020.10.09.331918

Figure Lengend Snippet: Generation of live APCs with fluorescent telomeres. ( a ) PNA-telomere probes were dissolved 1:50 in telomere loading buffer (80 mM KCl, 10 mM K2PO4, 4 mM NaCl, pH 7.2) and gently introduced on adherent APCs by rolling large glass beads (size: 400-600 nm). The beads are used to produce small holes in the APC plasma membranes that allow telomere labeling while preserving viable APCs (no cell fixative) needed for subsequent synapse studies with T cells. Arrow indicates beads. The beads are completely removed by washing with PBS prior to assays. ( b ) Absence of any residual glass beads from APCs was confirmed by field emission scanning electron microscopy (FESEM). Scale bar, 2 mm. ( c ) Identical telomere detection by IF-FISH (fixed cells) or glass bead-mediated telomere PNA probe delivery (live cells) in APCs. Four experiments. Scale bar, 20 μm. ( d ) Manders co-localization scores of experiments as in ( c ). Negative control, APCs not labelled with telomeres. ( e ) POT1 recruitment to telomeres detected by microbead-mediated telomere PNA probe delivery in human APCs, directly ex vivo . Scale bar, 10 mm. ( f ) Manders co-localization scores of experiments as in ( e ). Two experiments. Error bars indicate S.E.M. throughout.

Article Snippet: Human APCs were cultured with IL-4 (50 ng/mL) plus GM-CSF (10 ng/mL) for 48h, then transduced with POT1 (sc-403275-LAC) and TRF2 (sc-401289-LAC) lentiviral activation particles (both from Santa-Cruz Biotechnology).

Techniques: Clinical Proteomics, Labeling, Preserving, Electron Microscopy, Negative Control, Ex Vivo

Journal: bioRxiv

Article Title: Intercellular telomere transfer extends T cell lifespan

doi: 10.1101/2020.10.09.331918

Figure Lengend Snippet: ( a ) Representative metaphase spreads showing T cell chromosomes with APC-derived telomeres. T cells were incubated with cell-free supernatants from ionomycin-activated APCs that were live-labelled with TelC probes. Scale bar, 0.85 μm. ( b ) Metaphase spreads generated as in ( a ) were treated with 1unit T7 endonuclease for 30 min at 37 °C. The number of T cell chromosomes with APC telomeres before (black values) and after (red values) T7 endonuclease treatment is shown. Endonuclease treatment destroyed T cell chromosomes with APC telomeres while the total number of APC telomeres remained unaffected. ( c ) Presence of APC-derived telomeres in purified T cell plasma membranes vs T cell nuclei 24h after transfer of APC-free supernatants derived as in ( a ). ( d ) Quantification of APC telomere signal after T cell chromatin immunoprecipitation. Telomeres were extracted from Cy-3-PNA labelled APCs with ionomycin then transferred to T cells. The endogenous T cell telomere complex was immunoprecipitated (IP) 24h later with anti-POT1 and the Cy3-fluorescence of APC-derived telomeres quantified with a microplate reader. Control IP, T cell extracts precipitated with irrelevant IgG. Presence of APC-derived telomeres was confirmed by adding DNase directly to the POT1 IP for 10 min at RT prior to fluorescence reading. Two donors. Error bars indicate S.E.M. throughout.

Article Snippet: Human APCs were cultured with IL-4 (50 ng/mL) plus GM-CSF (10 ng/mL) for 48h, then transduced with POT1 (sc-403275-LAC) and TRF2 (sc-401289-LAC) lentiviral activation particles (both from Santa-Cruz Biotechnology).

Techniques: Derivative Assay, Incubation, Generated, Purification, Clinical Proteomics, Chromatin Immunoprecipitation, Immunoprecipitation, Fluorescence, Control

Journal: bioRxiv

Article Title: Intercellular telomere transfer extends T cell lifespan

doi: 10.1101/2020.10.09.331918

Figure Lengend Snippet: ( a ) Flow-cytometry quantification of telomere vesicles released by 10 7 APCs in culture media following the indicated treatments, with or without ionomycin for 18h. ( b ) Shelterin down-regulation in APCs following activation with ionomycin as in ( a ). Representative POT1 and TRF2 staining’s ( left ) and pooled data (n = 2; rights ) are shown. Each dot is an individual cell. Scale bar, 2 μm. ( c ) APCs donate shelterin-devoid telomeres. APCs were live-labelled with TelC telomere probes, activated with ionomycin, then analysed by IF to shelterin (POT1). Arrows indicate ‘shelterin-devoid’ telomeres released by APCs. Scale bar, 10 μm. Representative microscopy field ( left ) and pooled data from 30 fields ( right ) are shown. ( d ) APCs were transduced with mock or shelterin expressing (TRF2 + POT1) vectors, and labelled with BrdU. APCs were then activated 18h by ionomycin and telomere vesicles released by APCs were immunoprecipitated with anti-BrdU from cell-free supernatants. Representative blots ( bottom ) and data form three experiments ( top ) are shown. See also Extended Data Fig.9. ( e ) Telomere vesicles were analysed by IF to Rad51. Individual staining’s ( left ) and quantification from 42 microscopy fields is shown ( right ). Scale bars, 2 μm. ( f ) APCs were transfected with siCtrl or siRad51 for 36h, live-labelled with Cy3-PNA telomere probes then activated by ionomycin for 18h. The cell-free supernatants containing fluorescent APC-telomere vesicles were then transferred into T cells with FITC-PNA telomere labelling. There was no contact between APCs and T cells. APC-T cell telomere co-localization (Cy3 + FITC = yellow) in recipient T cell nuclei was quantified by confocal imaging 24h later. Representative images ( left ) and pooled data from 6 experiments ( right ) are shown. Arrowheads indicate APC-T cell telomere co-localization. Scale bar, 5 μm. In ( a , b, c, d, f ), Mann-Whitney test. *p<0.05 *** P < 0.001. Error bars indicate S.E.M. throughout.

Article Snippet: Human APCs were cultured with IL-4 (50 ng/mL) plus GM-CSF (10 ng/mL) for 48h, then transduced with POT1 (sc-403275-LAC) and TRF2 (sc-401289-LAC) lentiviral activation particles (both from Santa-Cruz Biotechnology).

Techniques: Flow Cytometry, Activation Assay, Microscopy, Transduction, Expressing, Immunoprecipitation, Transfection, Imaging, MANN-WHITNEY

Journal: bioRxiv

Article Title: Intercellular telomere transfer extends T cell lifespan

doi: 10.1101/2020.10.09.331918

Figure Lengend Snippet: ( a ) Representative images ( left ) and quantification ( right ) of TRF2 levels by immunofluorescence in APCs left either untreated, or pre-incubated with the proteasome inhibitor MG-132 (1mM) then conjugated with T cells for 24h. T=0, initial time at which conjugates are observed (20 min). Images were z-stacked and the raw telomere integrated fluorescence signals (AU, arbitrary units) are shown. 48 conjugates were analysed. Scale bar, 10 μm. ( b, top ) Validation of shelterin overexpression (TRF2 and POT1) by immunoblotting in APCs. Numbers indicate shelterin overexpression efficiency. ( b, bottom ) IF-FISH demonstrating recruitment of artificial shelterin factors (POT1 and TRF2) to telomeres in APCs transduced with lentiviral vectors and activated with ionomycin for 18h. The Pearson’s co-localization score for artificial POT1 and TRF2 with APC telomeres are shown. Scale bar, 5 μm. ( c ) Immunoblot analysis of TRF2 and POT1 following indicated siRNA treatment in human APCs. H2B served as loading control. The numbers indicate knock-down efficiency. Representative of 3 separate experiments. ( d ) Dot-blot analysis of telomeric DNA from the EVs released by APCs transfected as in ( c ) and activated with ionomycin for 18h. Quantification of the arbitrary units (AU) from 4 independent experiments ( top ) and representative dot-blot using TelC probe ( bottom ). In ( a ), Kruskal-Wallis with Dunn’s post-correction test; in ( d ), Mann-Whitney test. ** P < 0.01. Error bars indicate S.E.M. throughout.

Article Snippet: Human APCs were cultured with IL-4 (50 ng/mL) plus GM-CSF (10 ng/mL) for 48h, then transduced with POT1 (sc-403275-LAC) and TRF2 (sc-401289-LAC) lentiviral activation particles (both from Santa-Cruz Biotechnology).

Techniques: Immunofluorescence, Incubation, Fluorescence, Biomarker Discovery, Over Expression, Western Blot, Transduction, Control, Knockdown, Dot Blot, Transfection, MANN-WHITNEY