Journal: International Journal of Molecular Sciences

Article Title: Expression of Cellular and Extracellular TERRA , TERC and TERT in Hepatocellular Carcinoma

doi: 10.3390/ijms23116183

Figure Lengend Snippet: Tissue expression of TERC . ( a ) TERC relative quantification mean comparison between PT and HCC. Wilcoxon test was used. ( b ) TERC fold-change. Histograms represent R-values and bars upper and lower limits. Histograms are ordered by increasing R-values. The corresponding cases are listed on x axis. Upper and lower dot lines define the range of gene expression variation. ( c ) ROC curve analysis of TERC to discriminate HCC from normal tissue. AUC: area under the ROC curve’ CI: confidence interval.

Article Snippet: Then, 9 μL of the resulting cDNA was prepared for amplification in a 20 μL reaction volume containing 10 μL 2X ddPCR Supermix for probes (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and 1 μL 20X TaqMan (Thermo Fisher Scientific, Inc., Waltham, MA, USA) PCR probe assay ( TERT : assay ID Hs00972650_m1; TERC : assay ID Hs03454202_s1).

Techniques: Expressing, Quantitative Proteomics, Comparison, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Expression of Cellular and Extracellular TERRA , TERC and TERT in Hepatocellular Carcinoma

doi: 10.3390/ijms23116183

Figure Lengend Snippet: Plasma levels of TERC . ( a ) TERC levels in terms of copies/µL in plasma of control subjects (C) and HCC patients. Unpaired t -test was used. ( b ) ROC curve analysis of TERC to discriminate HCC from healthy individuals. ** p < 0.01; *** p < 0.001.

Article Snippet: Then, 9 μL of the resulting cDNA was prepared for amplification in a 20 μL reaction volume containing 10 μL 2X ddPCR Supermix for probes (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and 1 μL 20X TaqMan (Thermo Fisher Scientific, Inc., Waltham, MA, USA) PCR probe assay ( TERT : assay ID Hs00972650_m1; TERC : assay ID Hs03454202_s1).

Techniques: Clinical Proteomics, Control

Journal: International Journal of Molecular Sciences

Article Title: Expression of Cellular and Extracellular TERRA , TERC and TERT in Hepatocellular Carcinoma

doi: 10.3390/ijms23116183

Figure Lengend Snippet: Intra- and extracellular expression of TERRA and telomerase transcripts in HCC cell lines. The expression levels of TERRA ( a ), TERC ( b ) and TERT mRNA ( c ) were measured in HA22T/VGH and SKHep1C3 untreated, resistant to sorafenib (SR) and treated with 15 µM sorafenib. The levels of TERRA ( d ) and TERC ( e ) were measured in EVs from HA22T/VGH and SKHep1C3 cells untreated, resistant to sorafenib (SR) and treated with 15 µM sorafenib. As described in Materials and Methods, gene expression level was normalized to the number of particles released per each biological sample, respectively. The histograms represent the mean values of 2 experiments, bars are ± SEM. Unpaired two tailed t -test; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then, 9 μL of the resulting cDNA was prepared for amplification in a 20 μL reaction volume containing 10 μL 2X ddPCR Supermix for probes (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and 1 μL 20X TaqMan (Thermo Fisher Scientific, Inc., Waltham, MA, USA) PCR probe assay ( TERT : assay ID Hs00972650_m1; TERC : assay ID Hs03454202_s1).

Techniques: Expressing, Gene Expression, Two Tailed Test

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A ) Telomer length (TL) by flow-cytometry analysis of telomeric signal (FITC labelled PNA probe) showing increase in TL in HT1080-LT cells relative to HT1080 cells; plotted along x-axis in log scale. ( B ) TERT and TERC expression as expected was enhanced in HT1080-LT relative to HT1080 cells (normalised to GAPDH). [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( C ) Immuno-histochemical (IHC) staining of previously reported and well-characterised cells with short/long telomeres (fibrosarcoma HT1080 or the long-telomere version HT1080-LT cells in cell-isogenic background) hybridised with telomere-specific FITC-labelled PNA probe, counterstained with DAPI (left panel); telomeric signal was enhanced in HT1080-LT cells (quantified in right panel). [N=15] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( D ) Volcano plot of expressed genes in RNA-seq in HT1080 or HT1080-LT cells; x-axis has log-2 fold change for genes and the y-axis represents adjusted p values in log-10 scale. Key inflammation-related genes that have been marked in figure – most differentially expressed genes like IL1R1 , IL1B and IL10 were found to have higher expression in HT1080 cells compared to HT1080-LT cells. TERT which (as expected) is significantly lower in HT1080 cells compared to HT1080-LT cells have been also marked. Upregulated and downregulated genes listed in . ( E ) Gene Ontology (KEGG) for upregulated genes in HT1080 cells over HT1080-LT cells. ( F ) Gene Ontology (KEGG) for down-regulated genes in HT1080 cells over HT1080-LT cells. ( G ) Occupancy of TRF2 by ChIP-qPCR checked in HT1080 cells for cytokine-related genes. Enrichment was plotted relative to mock IgG (post normalisation to 1% input) [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( H ) TRF2 occupancy on the IL1R1 promoter in HT1080, MDAMB231, HEK293T or MRC5 cells by chromatin-immunoprecipitation (ChIP) followed by qPCR. Primers spanning +200 to –1000 bp of TSS used for TRF2 enrichment on the gene promoter; 3’UTR and –20 Kb upstream of TSS used as negative control for TRF2 binding; fold-change of occupancy was calculated over mock IgG after normalizing signal to 1% input. [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( I ) IL1R1 protein levels in HT1080 and HT1080-LT cells by western blot; GAPDH was used as loading control. ( J ) mRNA expression of hTERT and TERC (normalised to 18 S) in xenograft tumours in NOD-SCID mice injected with HT1080 or HT1080-LT cells. 18 S has been used as a normalizing control. [N=5] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( K ) hTERT mRNA expression in hTERT-inducible HT1080 cells on treatment with different concentrations of doxycycline [N=2] (left panel) and at different days following induction [N=3] (right panel). GAPDH was used for normalisation. Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( L ) Relative telomere length by qRT-PCR in MDAMB231 or MDAMD231-LT (long telomere) cells in three independent experiments. [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). Error bars correspond to SEM from independent experiments. Figure 1—figure supplement 2—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 1—figure supplement 2—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 1—figure supplement 2—source data 3. Original image files for western blot for .

Article Snippet: Recombinant DNA reagent , TERC shRNA , Santa-cruz , sc-106994-SH , Plasmid.

Techniques: Flow Cytometry, Expressing, Immunohistochemistry, RNA Sequencing, ChIP-qPCR, Chromatin Immunoprecipitation, Negative Control, Binding Assay, Western Blot, Control, Injection, Quantitative RT-PCR

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A, B ) NFKappaB activation in presence of IL1B (10 ng/ml) in HT1080 ( A ) or MDAMB231 ( B ) cells with or without TRF2 induction; activation signalling was confirmed through NFKappaB-Ser536 phosphorylation (normalised to total NFKappaB); ratio of Ser566-p/total NFKappaB plotted for respective blots from three independent replicates (right panels). [N=2]. ( C ) Expression of NFKappaB targets IL6, IL8 or TNF in presence/absence IL1A, IL1B, or TNF-a (10 ng/ml) for 24 hr in control (scrambled siRNA) or TRF2-low (TRF2 siRNA) conditions in HT1080 cells. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( D ) Expression of IL6, IL8, or TNF in control or TRF2-induced conditions on treatment with either IL1A or IL1B (10 ng/ml) for 24 hr in absence (left panel) or presence (right panel) of the IL1-receptor-antagonist IL1RA (20 ng/ml) in HT1080 cells. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( E ) NFkappaB/phosphor-Ser536-NFKappaB levels in xenograft tumours developed in NOD-SCID mice (control or doxycycline-induced TRF2 in HT1080 cells; N=6 mice in each group) by immuno-flow cytometry; mean fluorescence signal from individual tumours in control or TRF2-induced tumours plotted in adjacent graph; activation shown as ratio of pSer536-p-NFkappaB over total NFkappaB; significance was calculated using Wilcoxon’s non-parametric test. [N=6] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). Error bars correspond to SEM from independent experiments. Figure 6—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 6—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 6—source data 3. Original image files for western blot for .

Article Snippet: Recombinant DNA reagent , TERC shRNA , Santa-cruz , sc-106994-SH , Plasmid.

Techniques: Activation Assay, Phospho-proteomics, Expressing, Control, Flow Cytometry, Fluorescence, Western Blot

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A ) TNBC samples (N=6 each for long or short telomere samples) stained with EpCAM (far red) and then hybridised with telomere specific PNA probe (FITC). Mean telomeric signal was plotted in total cells. [N=6] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( B ) Telomere length was determined using the previously published algorithm Tel-Seq from sequenced genomes of TNBC samples (N=8 for TNBC-ST (short telomere) and N=6 for TNBC-LT (long telomere); and adjacent normal tissue from same patient). Table with sequencing related details of the normal/tumour samples has been provided. ( C ) Telomerase activity was measured from protein lysates across 64 TNBC samples (long or short telomeres) normalised to the telomerase activity of HT1080 cells for the same amount of protein; spearman correlation was calculated between RTL (relative telomere length) and telomerase activity. ( D ) mRNA expression of hTERT and TERC in six TNBC samples (long or short telomeres) normalised to the 18 S gene in respective samples. [N=6] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( E ) TNBC organoids (TNBO) formed from TNBO-ST (short telomere) and TNBO-LT (long telomere) samples. ( F ) Spearman correlation of, TERT , TERC, IL1R1 and cytokine-related gene expression and RTL (Relative telomere length) in 34 TNBC tissue samples with each other and RTL represented as heat map; gene expression was normalised to the18S gene; for telomere length the 36B4 single-copy gene was used for normalisation (as in ); fold change of TL was calculated with respect to TL in HT1080 cells. ( G ) Overall Survival for breast cancer patients (BRCA) with high IL1R1 mRNA expression [N=254] or low IL1R1 mRNA expression [N=709] from TCGA datasets (see for details) – graph tabulated using cBIOPORTAL with custom queries. Logrank p-value was found to be 0.0149. ( H ) Frequency of occurrence of samples with high / low IL1R1 mRNA expression in various annotated sub-types of breast cancer from TCGA (see for details). Error bars correspond to SEM from independent experiments. Figure 7—figure supplement 1—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values.

Article Snippet: Recombinant DNA reagent , TERC shRNA , Santa-cruz , sc-106994-SH , Plasmid.

Techniques: Staining, Sequencing, Activity Assay, Expressing, Gene Expression

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A ) Gene Ontology (GO) analysis of top 500 genes correlated with telomere elongation across 31 cancer types (as reported in ) Top 5 hits for ‘biological processes’ represented in descending order of enrichment; respective fold enrichments shown by individual bars (x-axis – top); the false discovery rate (FDR) shown by blue line. ( B ) Gating strategy for . ( C ) Gating strategy for CD 45+ve c gated ells and CD 11B+ve gated cells in TNBC. Percentage of CD 45+ve and CD11B+ve (in CD 45+ve population) was checked for TNBC-ST and TNBC-LT samples. [N=5] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). In the CD11B+ve (in CD 45+ve population), CD 206 (M2 macrophage marker) and CD 86 (M1 macrophage marker)+ve cells were quantified. Percentage of CD 206 high CD 86 low (M2 macrophages) and CD86 high Cd206 low (M1 macrophages) in the myeloid cell population (CD 45+ve CD 11B+ve) was plotted for three TNBC-ST and TNBC-LT samples each. [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( D ) TNBC tissue (N=6 each for long or short telomere samples) were tested for TERT and TERC expression. The Telomerase activity for these samples was estimated with HT1080 cell lysate as a reference. The normalised values (to the group average) were plotted in a heat map (left) for telomerase activity,TERT and TERC gene expression in the TNBC samples used in along with corresponding % TAM values (top left). The Spearman correlation matrix (top right) and p values (bottom) have been provided. ( E ) CD206 levels by immuno-flow cytometry in THP1 cells and macrophages differentiated from THP1 cells into M0 or M2 types (described in Materials and methods) and plotted along X-axis in log scale (top panel). Bright-field images at ×10 magnification for each cell stage shown in top right panel. ( F ) Different dosages of IL1RA was checked based on previously published reports and the minimal concentration where change in IL1R1 expression was seen (20 ng/ml) was used in other functional assays. [N=1]. ( G ) mRNA expression of IL1R1 was tested in MDAMB-231 cells after a 48 hr treatment with various reported G4-binding ligands. GAPDH was used as a normalizing control. [N=2]. Error bars correspond to SEM from independent experiments. Figure 8—figure supplement 1—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values.

Article Snippet: Recombinant DNA reagent , TERC shRNA , Santa-cruz , sc-106994-SH , Plasmid.

Techniques: Marker, Expressing, Activity Assay, Gene Expression, Flow Cytometry, Concentration Assay, Functional Assay, Binding Assay, Control

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet: ( A ) Key Immune marker expression (TAM markers, TAM related chemokines, T cell markers), TERT and TERC were plotted for fold change of transcript level expression (IL1R1 high / IL1R1 low) and adjusted p-values in breast cancer (BRCA) samples categorized as IL1R1 high or low from TCGA (see ). ( B ) Percentage TAM infiltration in TNBC tissue with short (TNBC-ST) or long (TNBC-LT) telomeres using markers CD11b and CD206 (CD11b + CD206 + cells shown in top right quadrant); quantification of TAM infiltration from individual TNBC-ST or TNBC-LT samples have been plotted. [N=9] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( C ) TNBC tissue slides stained with TAM-specific marker CD206 for macrophage infiltration within tissue (counterstained with DAPI); representative images for two independent long or short telomere TNBC tissues shown. ( D ) Labelled (red) M2 macrophages derived from THP1 cells incubated with triple-negative breast tissue-derived organoids (TNBO) for 12 hr (scheme in left panel). Infiltration of M2 macrophages in organoids shown as percentage of labelled (red) M2 macrophages in individual flow-cytometry plots (florescence signal (FL3) in x-axis in log scale); percentage infiltration from five TNBO-ST or five TNBO-LT samples shown in right panel. [N=5] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( E ) Labelled (red) M2 macrophage infiltration in presence/absence of receptor antagonist IL1RA (20 ng/ml) for tumour organoids with short (TNBO-ST) or long telomeres (TNBO-LT) in three replicates; percentage values for M2 infiltration plotted in right panel. Red florescence (FL3; y-axis in log scale) and respective percentage M2 infiltration values marked on top of individual flow cytometry plots. [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( F ) mRNA expression of IL1R1 was checked in presence of varying concentrations of G-quadruplex binding ligands (48 hr treatment) using TNBO. 18 S gene was used for normalisation. Following this, the ligand JD83 was used to check M2 infiltration in TNBO as in ( C, D ). The M2 infiltration has been plotted for control and JD 83 treated samples. Top panel- [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001).; bottom panel- [N=5] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). Error bars correspond to SEM from independent experiments. Figure 8—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values.

Article Snippet: Recombinant DNA reagent , TERC shRNA , Santa-cruz , sc-106994-SH , Plasmid.

Techniques: Marker, Expressing, Staining, Derivative Assay, Incubation, Flow Cytometry, Binding Assay, Control

Journal: eLife

Article Title: Telomere length sensitive regulation of interleukin receptor 1 type 1 (IL1R1) by the shelterin protein TRF2 modulates immune signalling in the tumour microenvironment

doi: 10.7554/eLife.95106

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , TERC shRNA , Santa-cruz , sc-106994-SH , Plasmid.

Techniques: Derivative Assay, Recombinant, Flow Cytometry, N-ChIP, Control, Plasmid Preparation, shRNA

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Patients with benign prostatic hyperplasia show shorter leukocyte telomere length but no association with telomerase gene polymorphisms in Han Chinese males

doi:

Figure Lengend Snippet: Summary of rs2736100, rs2736098 and rs12696304 genotypes in controls and BPH patients

Article Snippet: The TERC rs12696304 (C/G), and TERT rs2736100 (A/C) and rs2736098 (A/G) genotyping was performed using pre-designed TaqMan SNP genotyping assay kits on an ABI 7500 Life Tech (Applied Biosystems), as described [ 31 ].

Techniques:

Journal: Molecular Biology Reports

Article Title: Telomerase downregulation induces proapoptotic genes expression and initializes breast cancer cells apoptosis followed by DNA fragmentation in a cell type dependent manner

doi: 10.1007/s11033-013-2600-9

Figure Lengend Snippet: Telomerase activity in breast cancer (MCF7 and MDA-MB-231) and non-cancer (MCF10A) cells. (Typical TRAP-PAGE experiment) MCF7, MDA-MB-231 breast cancer and MCF10A non-cancer breast epithelial cells were subjected to telomerase activity assessment using a TRAP assay. For the analysis 0.2 μg of total protein extract was taken from breast cancer cells lysates. In order to detect the enzyme activity in MCF10A cells different quantities of total lysates were taken (up to 1.5 μg) due to a very low basal telomerase expression. The mean values of three independent experiments in duplicates were taken for significance calculation. Transfection reagent treated cells were used as control (100 %). The picture presents typical result out of seven separate experiments in duplicate. * p < 0.05. 1 , 2 –MCF7 control cells; 3 , 4 –carrier i.e. transfection reagent (Lipofectamine2000); 5 , 6 –TERT siRNA in MCF7 cells [100 nM]; 7 , 8 –TERT siRNA in MDA-MB-231 cells [100 nM]; 9 , 10 –mock siRNA [375 nM]; 11 , 12 –mock siRNA [100 nM]; 13 –HeLa cells extract (low standard); 14 , 15 –MCF10A extracts (1.5 and 0.2 μg); 16 HeLa cells extract (low standard) replication; 17 lysis buffer; 18 negative sample containing no lysis buffer; Internal control, internal standard to monitor PCR inhibition in every sample/lane

Article Snippet: 24 h after seeding in 12-well plates the culture medium was replaced by OPTIMEM (without serum and antibiotics) followed by transfection with specific pooled siRNA ( TERT from Dharmacon, ThermoFisher Scientific, IL, USA; TERC and TP1 from Santa Cruz Biotechnology, CA, USA).

Techniques: Activity Assay, TRAP Assay, Expressing, Transfection, Control, Lysis, Inhibition

Journal: Molecular Biology Reports

Article Title: Telomerase downregulation induces proapoptotic genes expression and initializes breast cancer cells apoptosis followed by DNA fragmentation in a cell type dependent manner

doi: 10.1007/s11033-013-2600-9

Figure Lengend Snippet: Efficiency of TERT downregulation at the protein level MCF7 and MDA-MB-231 cells were treated with 100 nM TERT siRNA ( lanes 3 and 4) and after 72 h Western blot analysis was performed using a polyclonal antibody (Rockland, USA, PA). C, control cells, treated with transfection reagent (Lipofectamine2000; lanes 1 and 2); 100, cells treated with TERT siRNA [100 nM]. The picture presents typical result out of three separate experiments in duplicate. * p < 0.05

Article Snippet: 24 h after seeding in 12-well plates the culture medium was replaced by OPTIMEM (without serum and antibiotics) followed by transfection with specific pooled siRNA ( TERT from Dharmacon, ThermoFisher Scientific, IL, USA; TERC and TP1 from Santa Cruz Biotechnology, CA, USA).

Techniques: Western Blot, Control, Transfection

Journal: Molecular Biology Reports

Article Title: Telomerase downregulation induces proapoptotic genes expression and initializes breast cancer cells apoptosis followed by DNA fragmentation in a cell type dependent manner

doi: 10.1007/s11033-013-2600-9

Figure Lengend Snippet: The effect of telomerase downregulation on the cell cycle of breast cancer cells Cells were treated with 100 nM specific siRNA or with camptothecin (as positive control) followed by propidium iodide labeling and flow cytometry analysis. The results were shown as relative apoptotic cell number. All experiments (three separate) were performed in triplicates, and error bars show the (±SEM) values. * p < 0.05. C control cells, TERT TERT targeting siRNA, Lipo transfection reagent (Lipofectamine2000), Cpt camptothecin [50 nM], * p < 0.05 (relative to control cells)

Article Snippet: 24 h after seeding in 12-well plates the culture medium was replaced by OPTIMEM (without serum and antibiotics) followed by transfection with specific pooled siRNA ( TERT from Dharmacon, ThermoFisher Scientific, IL, USA; TERC and TP1 from Santa Cruz Biotechnology, CA, USA).

Techniques: Positive Control, Labeling, Flow Cytometry, Control, Transfection

Journal: Molecular Biology Reports

Article Title: Telomerase downregulation induces proapoptotic genes expression and initializes breast cancer cells apoptosis followed by DNA fragmentation in a cell type dependent manner

doi: 10.1007/s11033-013-2600-9

Figure Lengend Snippet: The effect of telomerase downregulation on DNA fragmentation Cells were treated with 100 nM specific siRNA or with camptothecin (as positive control) and subjected to TUNEL analysis using terminal transferase and flow cytometry analysis. All experiments (three separate) were performed in triplicates, and error bars show the (±SEM) values. * p < 0.05. C control cells treated with transfection reagent (Lipofectamine2000), TERT TERT targeting siRNA, Cpt camptothecin [50 nM], Neg negative sample, without terminal transferase; * p < 0.05 (relative to control cells)

Article Snippet: 24 h after seeding in 12-well plates the culture medium was replaced by OPTIMEM (without serum and antibiotics) followed by transfection with specific pooled siRNA ( TERT from Dharmacon, ThermoFisher Scientific, IL, USA; TERC and TP1 from Santa Cruz Biotechnology, CA, USA).

Techniques: Positive Control, TUNEL Assay, Flow Cytometry, Control, Transfection

Journal: Molecular Biology Reports

Article Title: Telomerase downregulation induces proapoptotic genes expression and initializes breast cancer cells apoptosis followed by DNA fragmentation in a cell type dependent manner

doi: 10.1007/s11033-013-2600-9

Figure Lengend Snippet: The effect of telomerase downregulation on apoptosis in breast cancer MCF7 and MDA-MB-231 cells MCF7 ( open bars ) and MDA-MB-231 ( grey bars ) were treated with 100 nM specific siRNA or with camptothecin (as positive control) and subjected to Annexin V and propidium iodide labeling followed by flow cytometry analysis. All experiments (three separate) were performed in triplicates, and error bars show the (±SEM) values. * p < 0.05. C control cells treated with transfection reagent (Lipofectamine2000), TERT TERT targeting siRNA, Cpt camptothecin [50 nM]; * p < 0.05 (relative to control cells)

Article Snippet: 24 h after seeding in 12-well plates the culture medium was replaced by OPTIMEM (without serum and antibiotics) followed by transfection with specific pooled siRNA ( TERT from Dharmacon, ThermoFisher Scientific, IL, USA; TERC and TP1 from Santa Cruz Biotechnology, CA, USA).

Techniques: Positive Control, Labeling, Flow Cytometry, Control, Transfection

Journal: Molecular Biology Reports

Article Title: Telomerase downregulation induces proapoptotic genes expression and initializes breast cancer cells apoptosis followed by DNA fragmentation in a cell type dependent manner

doi: 10.1007/s11033-013-2600-9

Figure Lengend Snippet: The effect of telomerase silencing on expression of genes contributing to apoptosis control Cells were treated with 100 nM of TERT targeting siRNA and after 72 h the expression profile of proapoptotic ( a ) and antiapoptotic ( b ) genes in MCF7 and MDA-MB-231 cancer cells was assessed using qPCR. The experiment was performed in duplicates. The graphs represent the increase or decrease of the expression of the indicated genes relative to untreated control cells, (baseline at “0″ level)

Article Snippet: 24 h after seeding in 12-well plates the culture medium was replaced by OPTIMEM (without serum and antibiotics) followed by transfection with specific pooled siRNA ( TERT from Dharmacon, ThermoFisher Scientific, IL, USA; TERC and TP1 from Santa Cruz Biotechnology, CA, USA).

Techniques: Expressing, Control

Journal: Gut

Article Title: TERC polymorphisms are associated both with susceptibility to colorectal cancer and with longer telomeres

doi: 10.1136/gut.2011.239772

Figure Lengend Snippet: Telomerase RNA component expression (A) and telomere length (B) in HCT116 after transfection with telomerase RNA component constructs carrying the A and G alleles at rs2293607, compared with empty vector and untransfected cells. Error bars show SEM of experiments performed in triplicate.

Article Snippet: Based on pilot experiments that estimated the time of maximum TERC expression, we measured telomere lengths after 48 h. At the same time, we measured total TERC expression using Taqman real-time quantitative assay Hs03297287_s1 (Applied Biosystems), with SF3B1 expression as a control.

Techniques: Expressing, Transfection, Construct, Plasmid Preparation

Journal: Medicina

Article Title: Relative Leukocyte Telomere Length and Telomerase Complex Regulatory Markers Association with Leber’s Hereditary Optic Neuropathy

doi: 10.3390/medicina58091240

Figure Lengend Snippet: Hardy-Weinberg equilibrium analysis.

Article Snippet: RLTL measurement and TEP1 rs1760904, rs1713418, TERC rs12696304, and rs35073794 genotyping were conducted using real-time PCR according to the manufacturer’s recommendations using a Step One Plus real-time PCR system (Applied Biosystems, Foster City, CA, USA).

Techniques:

Journal: Medicina

Article Title: Relative Leukocyte Telomere Length and Telomerase Complex Regulatory Markers Association with Leber’s Hereditary Optic Neuropathy

doi: 10.3390/medicina58091240

Figure Lengend Snippet: Influence of TEP1 rs1760904, rs1713418, TERC rs12696304 on LHON development.

Article Snippet: RLTL measurement and TEP1 rs1760904, rs1713418, TERC rs12696304, and rs35073794 genotyping were conducted using real-time PCR according to the manufacturer’s recommendations using a Step One Plus real-time PCR system (Applied Biosystems, Foster City, CA, USA).

Techniques:

Journal: Medicina

Article Title: Relative Leukocyte Telomere Length and Telomerase Complex Regulatory Markers Association with Leber’s Hereditary Optic Neuropathy

doi: 10.3390/medicina58091240

Figure Lengend Snippet: Distribution of relative leukocyte telomere lengths by genotypes.

Article Snippet: RLTL measurement and TEP1 rs1760904, rs1713418, TERC rs12696304, and rs35073794 genotyping were conducted using real-time PCR according to the manufacturer’s recommendations using a Step One Plus real-time PCR system (Applied Biosystems, Foster City, CA, USA).

Techniques: Control