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Proteintech terf2

Internal validation of combined IRI diagnostic model. ( a ) Nomogram of Model Genes in the diagnostic model of IRI in the combined GEO dataset. ( b ) DCA plot of the Model Genes in the Combined GEO Dataset for the Diagnostic Model of IRI. ( c ) ROC curve of the RiskScore of the IRI diagnostic model from the combined GEO dataset. D-I. ROC curves for CDKN2B ( d ), ID1 ( e ), STAT3 ( f ), <t>TERF2</t> ( g ), TP53 ( h ), and ZNF277 ( i ) were analyzed between disease control groups using the combined GEO dataset. AUC < 0.9 indicated high accuracy, 0.7 < AUC0.9 indicated substantial accuracy, and 0.5 < AUC < 0.7 indicated lower accuracy.

Terf2, supplied by Proteintech, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/terf2/pmc12881650-296-16-18?v=Proteintech
Average 94 stars, based on 10 article reviews

terf2 - by Bioz Stars, 2026-08

94/100 stars

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1) Product Images from "The role of cellular senescence-related genes in ischemia–reperfusion injury and the identification of their biomarkers"

Article Title: The role of cellular senescence-related genes in ischemia–reperfusion injury and the identification of their biomarkers

Journal: Scientific Reports

doi: 10.1038/s41598-026-36076-2

Figure Legend Snippet: Internal validation of combined IRI diagnostic model. ( a ) Nomogram of Model Genes in the diagnostic model of IRI in the combined GEO dataset. ( b ) DCA plot of the Model Genes in the Combined GEO Dataset for the Diagnostic Model of IRI. ( c ) ROC curve of the RiskScore of the IRI diagnostic model from the combined GEO dataset. D-I. ROC curves for CDKN2B ( d ), ID1 ( e ), STAT3 ( f ), TERF2 ( g ), TP53 ( h ), and ZNF277 ( i ) were analyzed between disease control groups using the combined GEO dataset. AUC < 0.9 indicated high accuracy, 0.7 < AUC0.9 indicated substantial accuracy, and 0.5 < AUC < 0.7 indicated lower accuracy.

Techniques Used: Biomarker Discovery, Diagnostic Assay, Control

In vivo verification of targeted genes in IRI mice. ( a ) Workflow of experimental design. ( b - g ) mRNA levels of CDKN2B , ID1 , STAT3 , TP53 , TERF2 , and ZNF277 were assessed at different time points in mice model of IRI. ( h ) IHC of 5 model genes with AUC > 0.9. Bar = 50 μm. ( i - m ) Statistical plot of average optical density values of IHC for model genes ( CDKN2B , TP53 , TERF2 , STAT3 , and ID1 ). * p < 0.05, **p < 0.01, and *** p < 0.001.

Figure Legend Snippet: In vivo verification of targeted genes in IRI mice. ( a ) Workflow of experimental design. ( b - g ) mRNA levels of CDKN2B , ID1 , STAT3 , TP53 , TERF2 , and ZNF277 were assessed at different time points in mice model of IRI. ( h ) IHC of 5 model genes with AUC > 0.9. Bar = 50 μm. ( i - m ) Statistical plot of average optical density values of IHC for model genes ( CDKN2B , TP53 , TERF2 , STAT3 , and ID1 ). * p < 0.05, **p < 0.01, and *** p < 0.001.

Techniques Used: In Vivo

Image Search Results

13cisRA and Topotecan Modulate CB-HSPC Proliferation and DNA Damage Responses. a Proliferation of HSPCs in liquid media treated with topotecan (blue), 13cisRA (red), or left untreated (NT, black) after 5 days. Data are represented as median ± IQR, n = 6. Statistical significance was tested by paired t-test (**p < 0.01, ***p < 0.001, ****p < 0.0001). b Expression of retinoic acid receptors (RARA, RARB, RARG) after 7 days in HSPCs treated with topotecan (blue), 13cisRA (red), or left untreated (NT, black). Data are represented as median ± IQR, n = 6. Statistical significance was tested by paired t-test (*p < 0.05, ****p < 0.0001). ( c ) Expression of CDKN1A and CDKN2A (p14ARF) after 7 days expansion of HSPCs treated with topotecan (blue), 13cisRA (red), or left untreated (NT, black). Data are represented as median ± IQR, n = 6. Statistical significance was tested by paired t-test (**p < 0.01, ***p < 0.001). d Frequency and expression of γH2A.X after 7 days expansion of HSPCs treated with topotecan (blue), 13cisRA (red), or left untreated (NT, black). Data are represented as median ± IQR, n = 5. Statistical significance was tested by paired t-test (*p < 0.05). e Frequency and expression of HMGB1 after 7 days expansion of HSPCs treated with topotecan (blue), 13cisRA (red), or left untreated (NT, black). Data are represented as median ± IQR, n = 5. Statistical significance was tested by paired t-test (*p < 0.05, **p < 0.01). f Gene expression of TERF1, TERF2, and TINF2 after 7 days expansion of HSPCs treated with topotecan (blue), 13cisRA (red), or left untreated (NT, black). Data are represented as median ± IQR, n = 6. Statistical significance was tested by paired parametric test except for TINF2, which was tested by paired nonparametric test (*p < 0.05, **p < 0.01).

Journal: Scientific Reports

Article Title: High-Risk neuroblastoma therapeutics Topotecan and 13-cis-Retinoic acid modulate autophagy and induce DNA damage response in hematopoietic stem cells and monocytes

doi: 10.1038/s41598-025-19454-0

Figure Lengend Snippet: 13cisRA and Topotecan Modulate CB-HSPC Proliferation and DNA Damage Responses. a Proliferation of HSPCs in liquid media treated with topotecan (blue), 13cisRA (red), or left untreated (NT, black) after 5 days. Data are represented as median ± IQR, n = 6. Statistical significance was tested by paired t-test (**p < 0.01, ***p < 0.001, ****p < 0.0001). b Expression of retinoic acid receptors (RARA, RARB, RARG) after 7 days in HSPCs treated with topotecan (blue), 13cisRA (red), or left untreated (NT, black). Data are represented as median ± IQR, n = 6. Statistical significance was tested by paired t-test (*p < 0.05, ****p < 0.0001). ( c ) Expression of CDKN1A and CDKN2A (p14ARF) after 7 days expansion of HSPCs treated with topotecan (blue), 13cisRA (red), or left untreated (NT, black). Data are represented as median ± IQR, n = 6. Statistical significance was tested by paired t-test (**p < 0.01, ***p < 0.001). d Frequency and expression of γH2A.X after 7 days expansion of HSPCs treated with topotecan (blue), 13cisRA (red), or left untreated (NT, black). Data are represented as median ± IQR, n = 5. Statistical significance was tested by paired t-test (*p < 0.05). e Frequency and expression of HMGB1 after 7 days expansion of HSPCs treated with topotecan (blue), 13cisRA (red), or left untreated (NT, black). Data are represented as median ± IQR, n = 5. Statistical significance was tested by paired t-test (*p < 0.05, **p < 0.01). f Gene expression of TERF1, TERF2, and TINF2 after 7 days expansion of HSPCs treated with topotecan (blue), 13cisRA (red), or left untreated (NT, black). Data are represented as median ± IQR, n = 6. Statistical significance was tested by paired parametric test except for TINF2, which was tested by paired nonparametric test (*p < 0.05, **p < 0.01).

Article Snippet: The following TaqMan probes were used: CDKN1A (Hs00355782_m1), CDKN2A (p16 INK4A splicing isoform, Hs02902543_mH), CDKN2A (p14 ARF splicing isoform, Hs99999189_m1), TP53 (Hs01034249_m1), GATA1 (Hs01085823_m1), CEBPA (Hs00269972_s1), SPI1 (Hs02786711_m1), POT1 (Hs00209984_m1), TERF1 (Hs00819517_mH), TERF2 (Hs01030567_m1), TINF2 (Hs01554307_g1), RARA (Hs00940446_m1), RARB (Hs00977140_m1), RARG (Hs01559234_m1), ATG5 (Hs00169468_m1), ATG7 (Hs00893766_m1), SIRT1 (Hs01009006_m1), SIRT3 (Hs00953477_m1) and GAPDH (Hs02758991_g1).

Techniques: Expressing, Gene Expression

Journal: Scientific Reports

Article Title: The role of cellular senescence-related genes in ischemia–reperfusion injury and the identification of their biomarkers

doi: 10.1038/s41598-026-36076-2

Figure Lengend Snippet: Internal validation of combined IRI diagnostic model. ( a ) Nomogram of Model Genes in the diagnostic model of IRI in the combined GEO dataset. ( b ) DCA plot of the Model Genes in the Combined GEO Dataset for the Diagnostic Model of IRI. ( c ) ROC curve of the RiskScore of the IRI diagnostic model from the combined GEO dataset. D-I. ROC curves for CDKN2B ( d ), ID1 ( e ), STAT3 ( f ), TERF2 ( g ), TP53 ( h ), and ZNF277 ( i ) were analyzed between disease control groups using the combined GEO dataset. AUC < 0.9 indicated high accuracy, 0.7 < AUC0.9 indicated substantial accuracy, and 0.5 < AUC < 0.7 indicated lower accuracy.

Article Snippet: The slides were incubated with primary antibodies: anti-CDKN2B (1:50, Biodragon, China), TP53 (1:50, ProteinTech Group, China), TERF2 (1:50, ProteinTech Group, China), STAT3 (1:600, Servicebio, China), and ID1 (1:800, Servicebio, China).

Techniques: Biomarker Discovery, Diagnostic Assay, Control

Journal: Scientific Reports

Article Title: The role of cellular senescence-related genes in ischemia–reperfusion injury and the identification of their biomarkers

doi: 10.1038/s41598-026-36076-2

Figure Lengend Snippet: In vivo verification of targeted genes in IRI mice. ( a ) Workflow of experimental design. ( b - g ) mRNA levels of CDKN2B , ID1 , STAT3 , TP53 , TERF2 , and ZNF277 were assessed at different time points in mice model of IRI. ( h ) IHC of 5 model genes with AUC > 0.9. Bar = 50 μm. ( i - m ) Statistical plot of average optical density values of IHC for model genes ( CDKN2B , TP53 , TERF2 , STAT3 , and ID1 ). * p < 0.05, **p < 0.01, and *** p < 0.001.

Techniques: In Vivo

Elevated TERF2 expression in AML patients and its correlation with clinicopathological features. (A) Transcriptional expression of TERF2 in AML analyzed using TCGA database. (B) TERF2 mRNA levels quantified by qRT-PCR in PBMCs from 50 AML patients and 35 healthy donors. (C) UMAP of single cells from an AML patient ( GSE116256 ), single-cell profiling reveals distinct expression patterns of TERF2 across cellular subpopulations in AML. (D) Heatmap depicting the average expression level of TERF2 across distinct cell types within the AML sample. (E) Univariate Cox proportional hazards regression analysis comparing survival outcomes between high- and low-TERF2 expression groups. (F) Prognostic accuracy of TERF2 evaluated using ROC curve analysis. (G) The predictive performance of the novel risk stratification model was evaluated through time-dependent ROC curve analysis, with AUC values calculated at 1-, 3-, and 5-year intervals to quantify sensitivity and specificity in AML prognosis. ***, P<0.001; ****, P<0.0001. AML, acute myeloid leukemia; AUC, area under the curve; CI, confidence interval; FPR, false positive rate; HR, hazard ratio; PBMCs, peripheral blood mononuclear cells; qRT-PCR, quantitative reverse transcription polymerase chain reaction; ROC, receiver operating characteristic; TCGA, The Cancer Genome Atlas; TPM, transcripts per million; TPR, true positive rate; UMAP, uniform manifold approximation and projection.

Journal: Translational Cancer Research

Article Title: High TERF2 expression is associated with poor prognosis and its suppression attenuates progression in acute myeloid leukemia

doi: 10.21037/tcr-2025-1226

Figure Lengend Snippet: Elevated TERF2 expression in AML patients and its correlation with clinicopathological features. (A) Transcriptional expression of TERF2 in AML analyzed using TCGA database. (B) TERF2 mRNA levels quantified by qRT-PCR in PBMCs from 50 AML patients and 35 healthy donors. (C) UMAP of single cells from an AML patient ( GSE116256 ), single-cell profiling reveals distinct expression patterns of TERF2 across cellular subpopulations in AML. (D) Heatmap depicting the average expression level of TERF2 across distinct cell types within the AML sample. (E) Univariate Cox proportional hazards regression analysis comparing survival outcomes between high- and low-TERF2 expression groups. (F) Prognostic accuracy of TERF2 evaluated using ROC curve analysis. (G) The predictive performance of the novel risk stratification model was evaluated through time-dependent ROC curve analysis, with AUC values calculated at 1-, 3-, and 5-year intervals to quantify sensitivity and specificity in AML prognosis. ***, P<0.001; ****, P<0.0001. AML, acute myeloid leukemia; AUC, area under the curve; CI, confidence interval; FPR, false positive rate; HR, hazard ratio; PBMCs, peripheral blood mononuclear cells; qRT-PCR, quantitative reverse transcription polymerase chain reaction; ROC, receiver operating characteristic; TCGA, The Cancer Genome Atlas; TPM, transcripts per million; TPR, true positive rate; UMAP, uniform manifold approximation and projection.

Article Snippet: Primary antibodies targeting TERF2 (#13136, 1:500 dilution), CDK4 (#12790, 1:500), CDK6 (#1331, 1:500), and CDKN2A (#80772, 1:500) were commercially obtained from CST (Danvers, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription, Polymerase Chain Reaction

Downregulation of TERF2 suppresses AML cells viability and proliferation. (A) qRT-PCR assays analyzed TERF2 mRNA levels of NC or TERF2-knockdown MOLM13 cells and THP1 cells. (B) Western blot assays detected TERF2 protein levels of NC or TERF2-knockdown MOLM13 cells and THP1 cells. (C,D) CCK-8 assay showing the viability of NC or TERF2-knockdown MOLM13 cells (C) and THP1 cells (D). (E-H) Flow cytometry assay analyzed cell cycle in NC or TERF2-knockdown MOLM13 cells (E,F) and THP1 cells (G,H). **, P<0.01; ****, P<0.0001; ns, not significant. AML, acute myeloid leukemia; CCK-8, Cell Counting Kit-8; NC, negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

Journal: Translational Cancer Research

Article Title: High TERF2 expression is associated with poor prognosis and its suppression attenuates progression in acute myeloid leukemia

doi: 10.21037/tcr-2025-1226

Figure Lengend Snippet: Downregulation of TERF2 suppresses AML cells viability and proliferation. (A) qRT-PCR assays analyzed TERF2 mRNA levels of NC or TERF2-knockdown MOLM13 cells and THP1 cells. (B) Western blot assays detected TERF2 protein levels of NC or TERF2-knockdown MOLM13 cells and THP1 cells. (C,D) CCK-8 assay showing the viability of NC or TERF2-knockdown MOLM13 cells (C) and THP1 cells (D). (E-H) Flow cytometry assay analyzed cell cycle in NC or TERF2-knockdown MOLM13 cells (E,F) and THP1 cells (G,H). **, P<0.01; ****, P<0.0001; ns, not significant. AML, acute myeloid leukemia; CCK-8, Cell Counting Kit-8; NC, negative control; qRT-PCR, quantitative reverse transcription polymerase chain reaction.

Techniques: Quantitative RT-PCR, Knockdown, Western Blot, CCK-8 Assay, Flow Cytometry, Cell Counting, Negative Control, Reverse Transcription, Polymerase Chain Reaction

TERF2 deficiency promotes apoptosis in AML cells. (A-D) Flow cytometry assay analyzed apoptosis in NC or TERF2-knockdown MOLM13 cells (A,B) or THP1 cells (C,D). (E-H) Western blot detected cleaved caspase 3 levels of NC or TERF2-knockdown MOLM13 cells (E,F) or THP1 cells (G,H). ***, P<0.001; ****, P<0.0001. AML, acute myeloid leukemia; NC, negative control.

Journal: Translational Cancer Research

Article Title: High TERF2 expression is associated with poor prognosis and its suppression attenuates progression in acute myeloid leukemia

doi: 10.21037/tcr-2025-1226

Figure Lengend Snippet: TERF2 deficiency promotes apoptosis in AML cells. (A-D) Flow cytometry assay analyzed apoptosis in NC or TERF2-knockdown MOLM13 cells (A,B) or THP1 cells (C,D). (E-H) Western blot detected cleaved caspase 3 levels of NC or TERF2-knockdown MOLM13 cells (E,F) or THP1 cells (G,H). ***, P<0.001; ****, P<0.0001. AML, acute myeloid leukemia; NC, negative control.

Techniques: Flow Cytometry, Knockdown, Western Blot, Negative Control

TERF2 involved in cuproptosis through E2F pathway in AML. (A) Bar graphs illustrate the associations between TERF2 expression levels and gene hallmark sets in AML patients. (B) GSEA for AML patients with high TERF2 expression in TCGA database. (C-E) Western blot analysis of E2F1, CDK4, CDK6, CDKN2A levels in MOLM13 cells and NB4 cells with or without TERF2 silence. (F,G) MOLM13 (F) and NB4 (G) cells with TERF2 knockdown were treated with specified concentrations of ES-Cu (1:1 ratio) for 48 hours, followed by cell viability assessment using the CCK-8 assay. ***, P<0.001; ****, P<0.0001. AML, acute myeloid leukemia; CCK-8, Cell Counting Kit-8; ES-Cu, elesclomol-copper; GSEA, gene set enrichment analysis; IC 50 , half-maximal inhibitory concentration; TCGA, The Cancer Genome Atlas.

Journal: Translational Cancer Research

Article Title: High TERF2 expression is associated with poor prognosis and its suppression attenuates progression in acute myeloid leukemia

doi: 10.21037/tcr-2025-1226

Figure Lengend Snippet: TERF2 involved in cuproptosis through E2F pathway in AML. (A) Bar graphs illustrate the associations between TERF2 expression levels and gene hallmark sets in AML patients. (B) GSEA for AML patients with high TERF2 expression in TCGA database. (C-E) Western blot analysis of E2F1, CDK4, CDK6, CDKN2A levels in MOLM13 cells and NB4 cells with or without TERF2 silence. (F,G) MOLM13 (F) and NB4 (G) cells with TERF2 knockdown were treated with specified concentrations of ES-Cu (1:1 ratio) for 48 hours, followed by cell viability assessment using the CCK-8 assay. ***, P<0.001; ****, P<0.0001. AML, acute myeloid leukemia; CCK-8, Cell Counting Kit-8; ES-Cu, elesclomol-copper; GSEA, gene set enrichment analysis; IC 50 , half-maximal inhibitory concentration; TCGA, The Cancer Genome Atlas.

Techniques: Expressing, Western Blot, Knockdown, CCK-8 Assay, Cell Counting, Concentration Assay

Knockdown of TERF2 suppresses AML progression and enhances cuproptosis sensitivity in vivo . (A,B) Tumor growth was monitored by bioluminescence imaging in MOLM13-engrafted NSG mice (A) and the quantification of luciferase signals for all mice per group (B). (C) Kaplan-Meier analysis of survival of MOLM13-engrafted mice, log rank test. (D,E) Tumor growth was monitored by bioluminescence imaging in MOLM13-engrafted mice treated with elesclomol (D) and the quantification of luciferase signals for all mice per group (E). (F) Kaplan-Meier analysis of survival of MOLM13-engrafted mice treated with elesclomol, log rank test. **, P<0.01. AML, acute myeloid leukemia; NSG, NOD-SCID IL2rg.

Journal: Translational Cancer Research

Article Title: High TERF2 expression is associated with poor prognosis and its suppression attenuates progression in acute myeloid leukemia

doi: 10.21037/tcr-2025-1226

Figure Lengend Snippet: Knockdown of TERF2 suppresses AML progression and enhances cuproptosis sensitivity in vivo . (A,B) Tumor growth was monitored by bioluminescence imaging in MOLM13-engrafted NSG mice (A) and the quantification of luciferase signals for all mice per group (B). (C) Kaplan-Meier analysis of survival of MOLM13-engrafted mice, log rank test. (D,E) Tumor growth was monitored by bioluminescence imaging in MOLM13-engrafted mice treated with elesclomol (D) and the quantification of luciferase signals for all mice per group (E). (F) Kaplan-Meier analysis of survival of MOLM13-engrafted mice treated with elesclomol, log rank test. **, P<0.01. AML, acute myeloid leukemia; NSG, NOD-SCID IL2rg.

Techniques: Knockdown, In Vivo, Imaging, Luciferase

The binding of TRF2 outside of telomeric regions (non-telomeric) is dependent on telomere length. When telomeres shorten, the occupancy of TRF2 at telomeres diminishes, resulting in increased TRF2 binding at non-telomeric sites. This shift in TRF2 distribution triggers epigenetic modifications at promoters showing telomere-dependent gene regulation. Schematic reused from Supplementary Information in Mukherjee et al, eLife, 2025.

Journal: eLife

Article Title: Telomeres control human telomerase ( TERT ) expression through non-telomeric TRF2

doi: 10.7554/eLife.104045

Figure Lengend Snippet: The binding of TRF2 outside of telomeric regions (non-telomeric) is dependent on telomere length. When telomeres shorten, the occupancy of TRF2 at telomeres diminishes, resulting in increased TRF2 binding at non-telomeric sites. This shift in TRF2 distribution triggers epigenetic modifications at promoters showing telomere-dependent gene regulation. Schematic reused from Supplementary Information in Mukherjee et al, eLife, 2025.

Article Snippet: Recombinant DNA Reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Binding Assay

( A ) Telomere length in isogenic cancer cell lines with short telomeres (ST, in grey) or long telomeres (LT, in blue) namely, HT1080-ST/LT, MDA-MB-231-ST/LT, and HCT116- ST/LT (Telomere trimming-Cas9 and TERC knockdown) as determined by Flow cytometry (FACS). MDA-MB-231-ST/LT and HCT116-ST/LT Telomere trimming-Cas9 models were generated by telomerase-independent mode of telomere length (TL) alteration (See Methods). Relative fold change is shown in insets with FACS plots. ( B–C ) ChIP followed by qRT-PCR at the 0–300 bp TERT promoter (upstream of TSS) for TRF2 ( B ) and H3K27me3 ( C ) in respective ST/LT cells as mentioned in ( A ); occupancy normalised to respective IgG or total Histone H3 (for H3K27me3). qPCR on the GAPDH promoter was used as the negative control in all cases. ( D ) TERT mRNA expression by qRT-PCR in ST/LT cell line pairs as mentioned in ( A ) normalised to GAPDH or 18 S mRNA levels. Primers specific to 3'UTR for endogenous TERT were used for the HT1080-ST/LT system where telomerase was overexpressed for telomere elongation; primers for functional (reverse transcriptase domain) (exon7/8) and full-length (exon 15/16) transcript were used for all other systems. MDA-MB-231-ST/LT and HCT116-ST/LT Telomere trimming-Cas9 models have been analysed as paired samples in each biological replicates. Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. P-values are calculated by an unpaired t-test for all data except mRNA for MDA-MB-231-ST/LT and HCT116-ST/LT Telomere trimming-Cas9 models where paired t-test was performed. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 2—source data 1. Source data for all plots in , related to HT1080 ST/LT cell line model. Figure 2—source data 2. Source data for all plots in , related to MDA MB 231 ST/LT cell line model. Figure 2—source data 3. Source data for all plots in , related to HCT116 ST/LT Telomere trimming-Cas9 model. Figure 2—source data 4. Source data for all plots in , related to HCT116 ST/LT TERC knockdown model.

Journal: eLife

Article Title: Telomeres control human telomerase ( TERT ) expression through non-telomeric TRF2

doi: 10.7554/eLife.104045

Figure Lengend Snippet: ( A ) Telomere length in isogenic cancer cell lines with short telomeres (ST, in grey) or long telomeres (LT, in blue) namely, HT1080-ST/LT, MDA-MB-231-ST/LT, and HCT116- ST/LT (Telomere trimming-Cas9 and TERC knockdown) as determined by Flow cytometry (FACS). MDA-MB-231-ST/LT and HCT116-ST/LT Telomere trimming-Cas9 models were generated by telomerase-independent mode of telomere length (TL) alteration (See Methods). Relative fold change is shown in insets with FACS plots. ( B–C ) ChIP followed by qRT-PCR at the 0–300 bp TERT promoter (upstream of TSS) for TRF2 ( B ) and H3K27me3 ( C ) in respective ST/LT cells as mentioned in ( A ); occupancy normalised to respective IgG or total Histone H3 (for H3K27me3). qPCR on the GAPDH promoter was used as the negative control in all cases. ( D ) TERT mRNA expression by qRT-PCR in ST/LT cell line pairs as mentioned in ( A ) normalised to GAPDH or 18 S mRNA levels. Primers specific to 3'UTR for endogenous TERT were used for the HT1080-ST/LT system where telomerase was overexpressed for telomere elongation; primers for functional (reverse transcriptase domain) (exon7/8) and full-length (exon 15/16) transcript were used for all other systems. MDA-MB-231-ST/LT and HCT116-ST/LT Telomere trimming-Cas9 models have been analysed as paired samples in each biological replicates. Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. P-values are calculated by an unpaired t-test for all data except mRNA for MDA-MB-231-ST/LT and HCT116-ST/LT Telomere trimming-Cas9 models where paired t-test was performed. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 2—source data 1. Source data for all plots in , related to HT1080 ST/LT cell line model. Figure 2—source data 2. Source data for all plots in , related to MDA MB 231 ST/LT cell line model. Figure 2—source data 3. Source data for all plots in , related to HCT116 ST/LT Telomere trimming-Cas9 model. Figure 2—source data 4. Source data for all plots in , related to HCT116 ST/LT TERC knockdown model.

Article Snippet: Recombinant DNA Reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Knockdown, Flow Cytometry, Generated, Quantitative RT-PCR, Negative Control, Expressing, Functional Assay, Reverse Transcription

( A ) Scheme depicting CRISPR modified HEK293T cells with 1300 bp TERT promoter driving Gaussia luciferase ( Gaussia Luc) construct inserted at the CCR5 safe harbour locus. Scheme denotes ChIP primers used to study chromatin occupancy of 0–300 bp TERT promoter region inserted at the exogenous locus. ( B ) Relative fold change in telomere length in TERT promoter insert cells following telomere shortening determined by FACS; quantification in the right panel. ( C , D ) ChIP followed by qRT-PCR at the 0–300 bp TERT promoter (upstream of TSS) insert at CCR5 locus for TRF2 ( C ), and H3K27me3 ( D ) in ST/LT cells. Occupancy normalised to respective IgG or total histone H3 (for H3K27me3). ( E ) TERT promoter- Gaussia luciferase activity in ST cells over LT cells from inserted exogenous with TERT promoter. Reporter activity is presented as luminescence (arbitrary units, a.u.) normalised to respective total protein levels. Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. P-values are calculated by an unpaired t-test for all data except mRNA for MDA-MB-231-ST/LT and HCT116-ST/LT Telomere trimming-Cas9 models where paired t-test was performed. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 4—source data 1. Source data for all plots in .

Journal: eLife

Article Title: Telomeres control human telomerase ( TERT ) expression through non-telomeric TRF2

doi: 10.7554/eLife.104045

Figure Lengend Snippet: ( A ) Scheme depicting CRISPR modified HEK293T cells with 1300 bp TERT promoter driving Gaussia luciferase ( Gaussia Luc) construct inserted at the CCR5 safe harbour locus. Scheme denotes ChIP primers used to study chromatin occupancy of 0–300 bp TERT promoter region inserted at the exogenous locus. ( B ) Relative fold change in telomere length in TERT promoter insert cells following telomere shortening determined by FACS; quantification in the right panel. ( C , D ) ChIP followed by qRT-PCR at the 0–300 bp TERT promoter (upstream of TSS) insert at CCR5 locus for TRF2 ( C ), and H3K27me3 ( D ) in ST/LT cells. Occupancy normalised to respective IgG or total histone H3 (for H3K27me3). ( E ) TERT promoter- Gaussia luciferase activity in ST cells over LT cells from inserted exogenous with TERT promoter. Reporter activity is presented as luminescence (arbitrary units, a.u.) normalised to respective total protein levels. Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. P-values are calculated by an unpaired t-test for all data except mRNA for MDA-MB-231-ST/LT and HCT116-ST/LT Telomere trimming-Cas9 models where paired t-test was performed. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 4—source data 1. Source data for all plots in .

Article Snippet: Recombinant DNA Reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: CRISPR, Modification, Luciferase, Construct, Quantitative RT-PCR, Activity Assay

( A ) Scheme depicting the protocol followed for doxycycline (Dox) inducible TERT overexpression in HT1080 cells (Dox-HT1080). ++/-- denotes the presence/ absence of dox at the indicated day points. ( B ) Relative fold change in telomere length at Day 0,10, and 24 determined by FACS in Dox-HT1080 cells; quantification in right panel. ( C–F ) ChIP followed by qRT-PCR at the 0–300 bp TERT promoter (upstream of TSS) for TRF2 ( C ) REST ( D ) EZH2 ( E ) or H3K27me3 ( F ) in Dox-HT1080 cells at Day 0,10, and 24; occupancy normalised to respective IgG or total Histone H3 (for H3K27me3). ( G ) TERT mRNA expression by qRT-PCR using TERT -specific 3'UTR primers in Dox- HT1080 cells at day intervals (as indicated); normalised to GAPDH mRNA levels. Fold changes were calculated independently for each biological replicate, as the three conditions represent paired samples (uninduced, induced with doxycycline, and post-doxycycline withdrawal). Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. One-way ANOVA followed by post-hoc tests (Tukey’s HSD) was performed to compare means across time points in Figs C-G (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 5—source data 1. Source data for all plots in .

Journal: eLife

Article Title: Telomeres control human telomerase ( TERT ) expression through non-telomeric TRF2

doi: 10.7554/eLife.104045

Figure Lengend Snippet: ( A ) Scheme depicting the protocol followed for doxycycline (Dox) inducible TERT overexpression in HT1080 cells (Dox-HT1080). ++/-- denotes the presence/ absence of dox at the indicated day points. ( B ) Relative fold change in telomere length at Day 0,10, and 24 determined by FACS in Dox-HT1080 cells; quantification in right panel. ( C–F ) ChIP followed by qRT-PCR at the 0–300 bp TERT promoter (upstream of TSS) for TRF2 ( C ) REST ( D ) EZH2 ( E ) or H3K27me3 ( F ) in Dox-HT1080 cells at Day 0,10, and 24; occupancy normalised to respective IgG or total Histone H3 (for H3K27me3). ( G ) TERT mRNA expression by qRT-PCR using TERT -specific 3'UTR primers in Dox- HT1080 cells at day intervals (as indicated); normalised to GAPDH mRNA levels. Fold changes were calculated independently for each biological replicate, as the three conditions represent paired samples (uninduced, induced with doxycycline, and post-doxycycline withdrawal). Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. One-way ANOVA followed by post-hoc tests (Tukey’s HSD) was performed to compare means across time points in Figs C-G (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 5—source data 1. Source data for all plots in .

Article Snippet: Recombinant DNA Reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Over Expression, Quantitative RT-PCR, Expressing

( A ) Scheme depicting protocol followed for doxycycline (Dox) inducible TERT overexpression in MDA-MB-231 (Dox-MDA-MB-231). ++/-- denotes the presence/ absence of dox at the indicated day points. ( B ) Relative fold change in telomere length at Day 0,10, and 14 determined by Flow cytometry in Dox-MDA-MB-231; quantification in right panel. ( C–F ) ChIP followed by qRT-PCR at the 0–300 bp TERT promoter (upstream of TSS) for TRF2 ( C ) REST ( D ) EZH2 ( E ) or H3K27me3 ( F ) in Dox-MDA-MB-231 cells at Day 0,10, and 14; occupancy normalised to respective IgG or total Histone H3 (for H3K27me3). ( G ) TERT mRNA expression by qRT-PCR using TERT-specific 3'UTR primer in Dox- MDA-MB-231 cells at day intervals (as indicated); normalised to GAPDH mRNA levels. Fold changes were calculated independently for each biological replicate, as the three conditions represent paired samples (uninduced, induced with doxycycline, and post-doxycycline withdrawal). Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. One-way ANOVA followed by post-hoc tests (Tukey’s HSD) was performed to compare means across time points in Figs C-G (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 6—source data 1. Source data for all plots in .

Journal: eLife

Article Title: Telomeres control human telomerase ( TERT ) expression through non-telomeric TRF2

doi: 10.7554/eLife.104045

Figure Lengend Snippet: ( A ) Scheme depicting protocol followed for doxycycline (Dox) inducible TERT overexpression in MDA-MB-231 (Dox-MDA-MB-231). ++/-- denotes the presence/ absence of dox at the indicated day points. ( B ) Relative fold change in telomere length at Day 0,10, and 14 determined by Flow cytometry in Dox-MDA-MB-231; quantification in right panel. ( C–F ) ChIP followed by qRT-PCR at the 0–300 bp TERT promoter (upstream of TSS) for TRF2 ( C ) REST ( D ) EZH2 ( E ) or H3K27me3 ( F ) in Dox-MDA-MB-231 cells at Day 0,10, and 14; occupancy normalised to respective IgG or total Histone H3 (for H3K27me3). ( G ) TERT mRNA expression by qRT-PCR using TERT-specific 3'UTR primer in Dox- MDA-MB-231 cells at day intervals (as indicated); normalised to GAPDH mRNA levels. Fold changes were calculated independently for each biological replicate, as the three conditions represent paired samples (uninduced, induced with doxycycline, and post-doxycycline withdrawal). Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. One-way ANOVA followed by post-hoc tests (Tukey’s HSD) was performed to compare means across time points in Figs C-G (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 6—source data 1. Source data for all plots in .

Article Snippet: Recombinant DNA Reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Over Expression, Flow Cytometry, Quantitative RT-PCR, Expressing

( B ) Relative fold change in telomere length in xenograft samples determined by qRT- PCR-based telomere length detection method as reported earlier ( and ). Telomeric signal normalised over single copy gene, 36B4. ( C ) TERT (exon 15/16 full-length transcript) and hTERC mRNA expression in xenograft tissues by qRT-PCR; normalised to 18 S mRNA levels. ( D, E ) ChIP followed by qPCR at the 0–300 bp TERT promoter (upstream of TSS) for TRF2 ( D ) and H3K27me3 ( E ); occupancy normalised to respective IgG and total H3 (for H3K27me3). Error bars represent ± SDs across individual values of n=5 xenograft tumour samples in each group. P-values are calculated by unpaired t-test with Welch’s correction (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 7—source data 1. Source data for all plots in .

Journal: eLife

Article Title: Telomeres control human telomerase ( TERT ) expression through non-telomeric TRF2

doi: 10.7554/eLife.104045

Figure Lengend Snippet: ( B ) Relative fold change in telomere length in xenograft samples determined by qRT- PCR-based telomere length detection method as reported earlier ( and ). Telomeric signal normalised over single copy gene, 36B4. ( C ) TERT (exon 15/16 full-length transcript) and hTERC mRNA expression in xenograft tissues by qRT-PCR; normalised to 18 S mRNA levels. ( D, E ) ChIP followed by qPCR at the 0–300 bp TERT promoter (upstream of TSS) for TRF2 ( D ) and H3K27me3 ( E ); occupancy normalised to respective IgG and total H3 (for H3K27me3). Error bars represent ± SDs across individual values of n=5 xenograft tumour samples in each group. P-values are calculated by unpaired t-test with Welch’s correction (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 7—source data 1. Source data for all plots in .

Article Snippet: Recombinant DNA Reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Quantitative RT-PCR, Expressing

( A ) Scheme depicting CRISPR-modified HEK293T cells with 1300 bp TERT promoter with G>A substitution at –124 or –146 bp (upstream of TSS) driving Gaussia luciferase ( Gaussia Luc) construct at the CCR5 safe harbour locus. Scheme denotes ChIP primers used to study chromatin occupancy of 0–300 bp TERT promoter region inserted at the exogenous locus. ( B ) Relative fold change in telomere length in two independent pairs of cells with short or long telomeres containing TERT promoter G4 disrupting mutations (–124G>A or –146 G>A) at CCR5 locus TERT promoter insert as determined by Flow cytometry; quantification in right panel. ( C , D ) ChIP followed by qRT-PCR at the inserted TERT promoter (0–300 bp upstream of TSS) for TRF2 ( C ), and H3K27me3 ( D ) in pairs of cells generated with –124G>A or –146 G>A mutation with long/ short telomeres, along with WT promoter ST/LT pair (as in ). Occupancy normalised to respective IgG or total histone H3 (for H3K27me3). ( E ) TERT promoter- Gaussia luciferase activity in short or long telomere cells with - 124G>A and –146 G>A mutated TERT promoter sequence, along with WT promoter ST/LT pair (as in ). Reporter activity presented as luminescence (arbitrary units, a.u.) normalised to respective total protein levels. Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc test for all pairwise comparisons. For planned comparisons between each parental and short-telomere cell line, unpaired t-tests were used. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 8—source data 1. Source data for all plats in .

Journal: eLife

Article Title: Telomeres control human telomerase ( TERT ) expression through non-telomeric TRF2

doi: 10.7554/eLife.104045

Figure Lengend Snippet: ( A ) Scheme depicting CRISPR-modified HEK293T cells with 1300 bp TERT promoter with G>A substitution at –124 or –146 bp (upstream of TSS) driving Gaussia luciferase ( Gaussia Luc) construct at the CCR5 safe harbour locus. Scheme denotes ChIP primers used to study chromatin occupancy of 0–300 bp TERT promoter region inserted at the exogenous locus. ( B ) Relative fold change in telomere length in two independent pairs of cells with short or long telomeres containing TERT promoter G4 disrupting mutations (–124G>A or –146 G>A) at CCR5 locus TERT promoter insert as determined by Flow cytometry; quantification in right panel. ( C , D ) ChIP followed by qRT-PCR at the inserted TERT promoter (0–300 bp upstream of TSS) for TRF2 ( C ), and H3K27me3 ( D ) in pairs of cells generated with –124G>A or –146 G>A mutation with long/ short telomeres, along with WT promoter ST/LT pair (as in ). Occupancy normalised to respective IgG or total histone H3 (for H3K27me3). ( E ) TERT promoter- Gaussia luciferase activity in short or long telomere cells with - 124G>A and –146 G>A mutated TERT promoter sequence, along with WT promoter ST/LT pair (as in ). Reporter activity presented as luminescence (arbitrary units, a.u.) normalised to respective total protein levels. Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc test for all pairwise comparisons. For planned comparisons between each parental and short-telomere cell line, unpaired t-tests were used. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 8—source data 1. Source data for all plats in .

Article Snippet: Recombinant DNA Reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: CRISPR, Modification, Luciferase, Construct, Flow Cytometry, Quantitative RT-PCR, Generated, Mutagenesis, Activity Assay, Sequencing

( A ) TRF2 mRNA levels in HT1080 cells as TRF2 PTM variants are overexpressed under endogenous TRF2 silenced conditions using shRNA ( B ) telomerase reverse transcriptase (TERT) full-length mRNA transcript (exon 15/16) levels by RT-PCR in HT1080 cells upon overexpression of TRF2 PTM variants as depicted in ( A ) under endogenous TRF2 knockdown condition ( B ). ( C ) TRF2 protein induction with Dox treatment in HT1080, HCT116, and MDAMB 231 TRF2 inducible lentiviral stable cells, confirmed by Western blot analysis Mol. Wt. ladder used in HT1080 and MDAMB 231 is Puregene 4 colour Prestained Protein Ladder,10–180 kDa and that of HCT116 is G Biosciences PAGEmark Tricolour PLUS. ( D ) Dose-dependent Dox induction of TRF2-WT and R17H variant (Left graph) in stable inducible TRF2 HT1080 cells to check TERT full-length mRNA transcript (exon 15/16) levels by RT-PCR (right graph). ( E ) Purified TRF2 WT and TRF2 R17H protein from HEK 293T cells as developed by anti-TRF2 and anti-FLAG antibodies (left panel) and representative Coomassie Brilliant Blue (CBB) gel for protein purification protocol. The lower band in the anti-FLAG blot is of bead-bound FLAG peptide. ( F ) H3K27 trimethylation levels in in vitro histone methyltransferase assay with empty (TRF2 unbound) FLAG beads. Error bars represent ± SDs from the mean from two independent biological replicates. P-values are calculated by an unpaired t-test in 4B. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 9—figure supplement 1—source data 1. Source data of all plots in . Figure 9—figure supplement 1—source data 2. PDF file containing original western blot for indicating the relevant bands and treatment. Figure 9—figure supplement 1—source data 3. Original image files for western blot for . Figure 9—figure supplement 1—source data 4. PDF file containing original western blot for indicating the relevant bands. Figure 9—figure supplement 1—source data 5. Original image files for western blot for .

Journal: eLife

Article Title: Telomeres control human telomerase ( TERT ) expression through non-telomeric TRF2

doi: 10.7554/eLife.104045

Figure Lengend Snippet: ( A ) TRF2 mRNA levels in HT1080 cells as TRF2 PTM variants are overexpressed under endogenous TRF2 silenced conditions using shRNA ( B ) telomerase reverse transcriptase (TERT) full-length mRNA transcript (exon 15/16) levels by RT-PCR in HT1080 cells upon overexpression of TRF2 PTM variants as depicted in ( A ) under endogenous TRF2 knockdown condition ( B ). ( C ) TRF2 protein induction with Dox treatment in HT1080, HCT116, and MDAMB 231 TRF2 inducible lentiviral stable cells, confirmed by Western blot analysis Mol. Wt. ladder used in HT1080 and MDAMB 231 is Puregene 4 colour Prestained Protein Ladder,10–180 kDa and that of HCT116 is G Biosciences PAGEmark Tricolour PLUS. ( D ) Dose-dependent Dox induction of TRF2-WT and R17H variant (Left graph) in stable inducible TRF2 HT1080 cells to check TERT full-length mRNA transcript (exon 15/16) levels by RT-PCR (right graph). ( E ) Purified TRF2 WT and TRF2 R17H protein from HEK 293T cells as developed by anti-TRF2 and anti-FLAG antibodies (left panel) and representative Coomassie Brilliant Blue (CBB) gel for protein purification protocol. The lower band in the anti-FLAG blot is of bead-bound FLAG peptide. ( F ) H3K27 trimethylation levels in in vitro histone methyltransferase assay with empty (TRF2 unbound) FLAG beads. Error bars represent ± SDs from the mean from two independent biological replicates. P-values are calculated by an unpaired t-test in 4B. (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 9—figure supplement 1—source data 1. Source data of all plots in . Figure 9—figure supplement 1—source data 2. PDF file containing original western blot for indicating the relevant bands and treatment. Figure 9—figure supplement 1—source data 3. Original image files for western blot for . Figure 9—figure supplement 1—source data 4. PDF file containing original western blot for indicating the relevant bands. Figure 9—figure supplement 1—source data 5. Original image files for western blot for .

Article Snippet: Recombinant DNA Reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: shRNA, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Over Expression, Knockdown, Western Blot, Variant Assay, Purification, Protein Purification, In Vitro, HMT Assay

( A ) TERT full-length transcript (exon 15/16) mRNA expression levels by qRT-PCR; normalised to GAPDH mRNA levels upon stable doxycycline induction of wild-type (WT) TRF2 or R17H TRF2 mutants in HT1080, HCT116, or MDA-MB-231 cells. ( B ) TERT mRNA FISH upon TRF2 WT or TRF2 R17H overexpression (untransfected, UT as control) in HT1080 cells; quantification shown in right panel. FLAG-tagged TRF2 overexpression was confirmed by Immunofluorescence microscopy. ( C–F ) ChIP followed by qRT-PCR at the 0–300 bp TERT promoter (upstream of TSS) for FLAG-tagged TRF2 ( C ), REST ( D ), EZH2 ( E ), or H3K27me3 ( F ) in HT1080 cells upon expression of WT TRF2 or TRF2 R17H. Occupancy normalised to respective IgG and total Histone H3 (for H3K27me3); qRT-PCR on the GAPDH promoter was used as the negative control in all cases. ( G ) In vitro methyltransferase activity of the reconstituted PRC2 complex resulting in H3K27 trimethylation in the presence or absence of TRF2 WT or TRF2 R17H protein. Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. Unpaired t-tests were conducted to assess the significance of each condition individually within the same cell line, and to compare the two conditions TRF2-WT or TRF2-R17H in ( A ); one-way ANOVA followed by post-hoc tests (Tukey’s HSD) was performed to compare means across the three conditions in ( B ); P-values are calculated by unpaired t-test in ( C–F ); and two-way ANOVA followed by post-hoc tests (Tukey’s HSD) in ( G ). (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 9—source data 1. Source data of all plots in , except for . Figure 9—source data 2. Source data of TERT mRNA FISH quantification in .

Journal: eLife

Article Title: Telomeres control human telomerase ( TERT ) expression through non-telomeric TRF2

doi: 10.7554/eLife.104045

Figure Lengend Snippet: ( A ) TERT full-length transcript (exon 15/16) mRNA expression levels by qRT-PCR; normalised to GAPDH mRNA levels upon stable doxycycline induction of wild-type (WT) TRF2 or R17H TRF2 mutants in HT1080, HCT116, or MDA-MB-231 cells. ( B ) TERT mRNA FISH upon TRF2 WT or TRF2 R17H overexpression (untransfected, UT as control) in HT1080 cells; quantification shown in right panel. FLAG-tagged TRF2 overexpression was confirmed by Immunofluorescence microscopy. ( C–F ) ChIP followed by qRT-PCR at the 0–300 bp TERT promoter (upstream of TSS) for FLAG-tagged TRF2 ( C ), REST ( D ), EZH2 ( E ), or H3K27me3 ( F ) in HT1080 cells upon expression of WT TRF2 or TRF2 R17H. Occupancy normalised to respective IgG and total Histone H3 (for H3K27me3); qRT-PCR on the GAPDH promoter was used as the negative control in all cases. ( G ) In vitro methyltransferase activity of the reconstituted PRC2 complex resulting in H3K27 trimethylation in the presence or absence of TRF2 WT or TRF2 R17H protein. Error bars represent ± SDs from the mean of three independent biological replicates of each experiment. Unpaired t-tests were conducted to assess the significance of each condition individually within the same cell line, and to compare the two conditions TRF2-WT or TRF2-R17H in ( A ); one-way ANOVA followed by post-hoc tests (Tukey’s HSD) was performed to compare means across the three conditions in ( B ); P-values are calculated by unpaired t-test in ( C–F ); and two-way ANOVA followed by post-hoc tests (Tukey’s HSD) in ( G ). (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 9—source data 1. Source data of all plots in , except for . Figure 9—source data 2. Source data of TERT mRNA FISH quantification in .

Article Snippet: Recombinant DNA Reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Control, Immunofluorescence, Microscopy, Negative Control, In Vitro, Activity Assay

( A ) Scheme showing generation of induced pluripotent stem cells (iPSCs) from foreskin fibroblast (FS Fibroblast) cells by overexpressing Yamanaka factors (Oct4, Sox2, Klf4, Myc). ( B ) Characterization of iPSCs (Upper panel, bright field image) generated from FS Fibroblast cells by immunofluorescence using Oct-4, SSEA-4, Sox-2, and TRA-1–60 antibodies as stemness markers. ( C ) mRNA levels for TERT (full-length exon 15/16 transcript), TERC (RNA component), and stemness marker genes Nanog, Klf4 in FS fibroblast and derived iPSC, analysed in pairs in each biological replicates. ( D ) Telomerase activity in FS Fibroblast cells and derived iPSCs determined using telomerase-repeat-amplification-protocol (TRAP) followed by ELISA (see Methods). ( E ) Relative fold change in telomere length in primary FS Fibroblast cells and derived iPSC, determined by qPCR-based telomere length detection method. ( F–I ) ChIP followed by qPCR at the 0–300 bp TERT promoter (upstream of TSS) for TRF2 ( F ), REST ( G ), EZH2 ( H ), and H3K27me3 ( I ) up to 300 bp upstream of transcription start site (TSS); occupancy normalized to respective IgG or total Histone H3 (for H3K27me3). qPCR on the GAPDH promoter was used as a negative control in all cases. All error bars represent ± SDs from the mean of two independent biological replicates of each experiment. P-values are calculated by an unpaired t-test (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 10—source data 1. Source data of all plots in . Figure 10—source data 2. Original images of iPSC IF in .

Journal: eLife

Article Title: Telomeres control human telomerase ( TERT ) expression through non-telomeric TRF2

doi: 10.7554/eLife.104045

Figure Lengend Snippet: ( A ) Scheme showing generation of induced pluripotent stem cells (iPSCs) from foreskin fibroblast (FS Fibroblast) cells by overexpressing Yamanaka factors (Oct4, Sox2, Klf4, Myc). ( B ) Characterization of iPSCs (Upper panel, bright field image) generated from FS Fibroblast cells by immunofluorescence using Oct-4, SSEA-4, Sox-2, and TRA-1–60 antibodies as stemness markers. ( C ) mRNA levels for TERT (full-length exon 15/16 transcript), TERC (RNA component), and stemness marker genes Nanog, Klf4 in FS fibroblast and derived iPSC, analysed in pairs in each biological replicates. ( D ) Telomerase activity in FS Fibroblast cells and derived iPSCs determined using telomerase-repeat-amplification-protocol (TRAP) followed by ELISA (see Methods). ( E ) Relative fold change in telomere length in primary FS Fibroblast cells and derived iPSC, determined by qPCR-based telomere length detection method. ( F–I ) ChIP followed by qPCR at the 0–300 bp TERT promoter (upstream of TSS) for TRF2 ( F ), REST ( G ), EZH2 ( H ), and H3K27me3 ( I ) up to 300 bp upstream of transcription start site (TSS); occupancy normalized to respective IgG or total Histone H3 (for H3K27me3). qPCR on the GAPDH promoter was used as a negative control in all cases. All error bars represent ± SDs from the mean of two independent biological replicates of each experiment. P-values are calculated by an unpaired t-test (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 10—source data 1. Source data of all plots in . Figure 10—source data 2. Original images of iPSC IF in .

Article Snippet: Recombinant DNA Reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Generated, Immunofluorescence, Marker, Derivative Assay, Activity Assay, Amplification, Enzyme-linked Immunosorbent Assay, Negative Control

( A ) Scheme depicting generation of induced pluripotent stem cells (iPSCs) with shortened telomeres, (iPSC-ST) using telomere-specific sgRNA-guided CRISPR-Cas9 to trim telomeres. ( B ) Relative fold change in telomere length in iPSC-ST cells with respect to unaltered iPSC, determined by qPCR-based telomere length detection method. ( C–D ) ChIP followed by qPCR at the 0–300 bp TERT promoter (upstream of TSS) in iPSC-ST cells in comparison to unaltered iPSC, for TRF2 ( L ) and H3K27me3 ( M ); occupancy normalized to respective IgG or total Histone H3 (for H3K27me3). qPCR on the GAPDH promoter was used as a negative control in all cases. ( E ) mRNA levels for TERT (full-length exon 15/16 transcript), TERC (RNA component), and TRF2 in iPSC-ST over unaltered iPSC. All error bars represent ± SDs from the mean of 2 independent biological replicates of each experiment. p values are calculated by unpaired t-test (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 11—source data 1. Source data of all plots in .

Journal: eLife

Article Title: Telomeres control human telomerase ( TERT ) expression through non-telomeric TRF2

doi: 10.7554/eLife.104045

Figure Lengend Snippet: ( A ) Scheme depicting generation of induced pluripotent stem cells (iPSCs) with shortened telomeres, (iPSC-ST) using telomere-specific sgRNA-guided CRISPR-Cas9 to trim telomeres. ( B ) Relative fold change in telomere length in iPSC-ST cells with respect to unaltered iPSC, determined by qPCR-based telomere length detection method. ( C–D ) ChIP followed by qPCR at the 0–300 bp TERT promoter (upstream of TSS) in iPSC-ST cells in comparison to unaltered iPSC, for TRF2 ( L ) and H3K27me3 ( M ); occupancy normalized to respective IgG or total Histone H3 (for H3K27me3). qPCR on the GAPDH promoter was used as a negative control in all cases. ( E ) mRNA levels for TERT (full-length exon 15/16 transcript), TERC (RNA component), and TRF2 in iPSC-ST over unaltered iPSC. All error bars represent ± SDs from the mean of 2 independent biological replicates of each experiment. p values are calculated by unpaired t-test (*p<0.05, **p<0.01, ***p<0.005, ****p<0.0001). Figure 11—source data 1. Source data of all plots in .

Article Snippet: Recombinant DNA Reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: CRISPR, Comparison, Negative Control

Illustration of the telomere-dependent epigenetic modification of chromatin structure at the TERT promoter, resulting in upregulation or downregulation of TERT expression due to altered non-telomeric TRF2 binding at the promoter. Relatively long telomeres cells (top panel) have lower TRF2 binding at the TERT promoter, promoting permissive chromatin and upregulation of TERT transcription. Conversely, shorter telomere cells (bottom panel) with increased TRF2 binding at the TERT promoter recruit more REST-PRC2 epigenetic complex causing increased repressor histone H3K27 trimethylation deposition. This leads to a more closed chromatin state at the TERT promoter, suppressing its transcription.

Journal: eLife

Article Title: Telomeres control human telomerase ( TERT ) expression through non-telomeric TRF2

doi: 10.7554/eLife.104045

Figure Lengend Snippet: Illustration of the telomere-dependent epigenetic modification of chromatin structure at the TERT promoter, resulting in upregulation or downregulation of TERT expression due to altered non-telomeric TRF2 binding at the promoter. Relatively long telomeres cells (top panel) have lower TRF2 binding at the TERT promoter, promoting permissive chromatin and upregulation of TERT transcription. Conversely, shorter telomere cells (bottom panel) with increased TRF2 binding at the TERT promoter recruit more REST-PRC2 epigenetic complex causing increased repressor histone H3K27 trimethylation deposition. This leads to a more closed chromatin state at the TERT promoter, suppressing its transcription.

Article Snippet: Recombinant DNA Reagent , TRF2 shRNA , origene , TL308880 , Plasmid.

Techniques: Modification, Expressing, Binding Assay

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