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: 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: 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: Subsequent incubation with the appropriate horseradish peroxidase-conjugated goat anti-rabbit POT1, TRF1, TRF2, TIN2, TRF2IP, GAPDH (cat. no. SP 9001) and goat anti-mouse TPP1 (cat. no. SP9002) secondary antibodies (1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was performed for 2 h at room temperature.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation, Control, Western Blot, Binding 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: 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: To evaluate Pot1 expression levels in hematopoietic stem/progenitor cells, BM Lin + , Lin − , LSK150 − , and LSKCD150 + cells (1 × 10 5 cells) were lysed and subjected to SDS–PAGE and immunoblotting using antibodies against POT1 polyclonal Ab (Santa Cruz Biotechnology, Inc.) (1:1000).

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: 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: To evaluate Pot1 expression levels in hematopoietic stem/progenitor cells, BM Lin + , Lin − , LSK150 − , and LSKCD150 + cells (1 × 10 5 cells) were lysed and subjected to SDS–PAGE and immunoblotting using antibodies against POT1 polyclonal Ab (Santa Cruz Biotechnology, Inc.) (1:1000).

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: POT1 prevents DDR in human HSC. a Expression of POT1 in hCB CD34 + cells and LT-HSCs (Lin – CD34 + CD38 − CD45RA − CD90 + CD49f + ). b hCB-derived LT-HSCs were cultured with MTM-POT1. After 10 days of culture, cells were isolated and re-cultured in methylcellulose medium (200 cells per dish). The number of CFU-C and HPP-CFC (>1.0 mm, >2.0 mm) are shown. Data are expressed as the mean ± SD ( n = 3, * p < 0.01 by t -test). Representative data from three independent experiments are shown. c , d hCB LT-HSCs were cultured for 10 days with control MTM protein or MTM-POT1. After 10 days of culture, LT-HSCs were re-isolated and number of TIF was examined. c Immunocytochemical staining of TRF1 ( green ), 53BP1 ( red ), and TOTO3 ( blue ). Scale bar , 2 μm ( left ). Frequencies of TIFs after 10 days of culture ( right ). Data are expressed as the mean ± SD ( n = 100: control, n = 100: MTM-POT1, * p < 0.01 by t -test). Representative data from 2 independent experiments are shown. d Immunocytochemical staining of TRF1 ( green ), RPA32 ( red ), and TOTO3 ( blue ). Scale bar , 2 μm ( left ). Frequencies of TIFs after 10 days of culture ( right ). Data are expressed as the mean ± SD ( n = 110–120: control, n = 110: MTM-POT1, * p < 0.01 by t -test). Representative data from two independent experiments are shown

Article Snippet: To evaluate Pot1 expression levels in hematopoietic stem/progenitor cells, BM Lin + , Lin − , LSK150 − , and LSKCD150 + cells (1 × 10 5 cells) were lysed and subjected to SDS–PAGE and immunoblotting using antibodies against POT1 polyclonal Ab (Santa Cruz Biotechnology, Inc.) (1:1000).

Techniques: Expressing, Derivative Assay, Cell Culture, Isolation, Control, 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: POT1 maintains self-renewal activity of human HSCs. hCB-derived LT-HSCs (200 cells per well) were cultured for 10 days with or without MTM-POT1. a Number of total cells ( left ) and Lin – CD34 + CD38 − CD45RA − CD90 + cells on day 7 and 10 of culture. Data are expressed as the mean ± SD ( n = 10, * p < 0.01 by t -test). b Representative FACS profiles of CD90 and CD45RA in Lin – CD34 + CD38 – CD49f + cells ( left ) and the expression of CD49f in the Lin – CD34 + CD38 − CD45RA − CD90 + fraction ( right ). c Percentage of Annexin V + PI + apoptotic cells in Lin – CD34 + CD38 − CD45RA − CD90 + cells on day 7 and 10 of culture. Data are expressed as the mean ± SD ( n = 11: day 7, n = 9: day 10). d LT-HSCs isolated from hBM (200 cells per well) were cultured for 10 days with or without MTM-POT1 (50 ng ml –1 ). Number of total cells, CD34 + CD38 − cells, and LT-HSCs after culture are shown. Data are expressed as the mean ± SD ( n = 3, * p < 0.01, ** p < 0.05 by t -test). Representative data from two independent experiments are shown. e Result of limiting dilution 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 = 5). The data point with zero negative response at dose 20,000 is represented by a downward-pointing triangle. Estimated numbers of repopulating cells are also shown

Article Snippet: To evaluate Pot1 expression levels in hematopoietic stem/progenitor cells, BM Lin + , Lin − , LSK150 − , and LSKCD150 + cells (1 × 10 5 cells) were lysed and subjected to SDS–PAGE and immunoblotting using antibodies against POT1 polyclonal Ab (Santa Cruz Biotechnology, Inc.) (1:1000).

Techniques: Activity Assay, Derivative Assay, Cell Culture, Expressing, Isolation