Journal: Communications Biology

Article Title: BLM and FANCJ role in the response to G-quadruplex-dependent telomeric replicative stress

doi: 10.1038/s42003-025-09367-z

Figure Lengend Snippet: a The diagram shows the workflow on a timeline: on the day after cell seeding, cells were treated with RHPS4 (0.2 and/or 0.5 μM). At 48 h after treatment, cells were again treated with RHPS4 (0.2 and/or 0.5 μM) or harvested and counted. At 72 h after the first treatment, the cell viability was checked, and finally at 96 h after the first treatment, the cells were harvested and processed. b Representative images showing U251MG cells stained by immunofluorescence using anti-BLM (red signals), and anti-TRF1 or anti-TRF2 (green signals) antibodies. Merged images allow visualization of colocalizing dots (yellow signals). Yellow arrows indicate BLM and TRF1-TRF2 colocalizations. c Quantification of the colocalizations between BLM and TRF1 or TRF2 proteins in U251MG cell line upon RHPS4 treatment (0.2 and 0.5 μM). d , e Sensitivity of U251MG and BLM −/− cell lines to RHPS4 concentrations ranging from 0.01 to 2 μM (0.01; 0.125; 0.25; 0.5; 1; 2 μM), evaluated 96 h after the first treatment. Mitomycin C (MMC) was used as a positive control at concentrations of 0.1; 0.5; 1; 2; 5 μg/ml. The Sulforhodamine B (SRB) cytotoxicity assay showed that RHPS4 sensitivity was unchanged in U251MG and BLM −/− (IC50 was 0.56 μM in both cell lines). f Short-term cell proliferation in untreated cells, U251MG and BLM −/− , and in RHPS4-treated cells (0.5 μM) as evaluated 48 and 96 h after the first treatment. g Long-term cell proliferation in U251MG and BLM −/− untreated and RHPS4-treated cells (0.5 μM). Scale bars represent 5 μm. * p < 0.05, ** p < 0.01 (two-way ANOVA; n = 3). Error bars denote the standard deviation of the mean.

Article Snippet: The slides were then incubated in blocking buffer (1% BSA dissolved in 1X PBS [w/v]) and then incubated overnight (ON) at 4 °C with the primary antibodies (rabbit polyclonal anti-BLM (#A300-110A, Bethyl) (1:100); mouse monoclonal anti-TRF1 (4E4 clone, GTX70304, GeneTex) (1:20) or mouse monoclonal anti-TRF2 (9F10 clone, sc-47693, Santa Cruz Biotechnology) (1:100)).

Techniques: Staining, Immunofluorescence, Positive Control, Cytotoxicity Assay, Standard Deviation

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: The binding of TRF2 outside of telomeric regions (non-telomeric) is dependent on telomere length. When telomeres shorten, the presence 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. Described in .

Article Snippet: Antibody , TRF2 (mouse monoclonal) , Millipore , (Millipore 4A794)- , Flow cytometry (1:500).

Techniques: Binding Assay

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: Antibody , TRF2 (mouse monoclonal) , Millipore , (Millipore 4A794)- , Flow cytometry (1:500).

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 ) Occupancy of TRF2 was checked by ChIP-qPCR on the IL1R1 promoter spanning +200 to –1000 bp of TSS was reduced ex vivo HT1080 cells with long telomeres (HL1080-LT) relative to ones with short telomeres (HT1080) cells (N=3 in each case); IL1R1-3’UTR or a region 20 kb upstream were used as negative controls for TRF2 binding. Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( B ) mRNA expression of IL1R1 in ex vivo HT1080 or HT1080-LT cells; GAPDH was used for normalisation [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 ) IL1R1 protein expression was checked by immuno-flow cytometry in HT1080 or HT1080-LT cells in three independent replicates; Mean IL1R1 expression has been plotted along the X-axis in log scale (right 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). ( D ) IL1R1 levels by immunofluorescence microscopy in HT1080 or HT1080-LT cells; cells were stained with DAPI for marking the cell nucleus; IL1R1 levels from 25 individual cells shown in graph (right panel). [N=25] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( E ) Telomere length of the xenograft tumours made in NOD SCID mice using HT1080 cells with long telomeres (HL1080-LT) and short telomeres (HT1080) was checked by flow-cytometry and was higher in HT1080-LT xenograft tumours. Following this, IL1R1 expression was checked by flow cytometry for these samples. Mean Telomeric signal and Mean IL1R1 expression has been plotted respectively (panels below) [N=5] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( F ) Occupancy of TRF2 by ChIP-qPCR at the IL1R1 promoter spanning +200 to –1000 bp of TSS was reduced in xenograft tumours made from HT1080 cells with long telomeres (HL1080-LT) relative to ones with short telomeres (HT1080) cells (N=3 in each case; IL1R1-3’UTR or a region 20 kb upstream were used as negative controls for TRF2 binding). [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( G ) mRNA expression of IL1R1 in xenograft tumours (HT1080 or HT1080-LT cells); 18 S was used for normalisation. [N=5] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( H ) Telomere length by flow-cytometry of telomeric signal in HT1080 cells (hTERT-inducible stable line) following 0, 1, 6, 12, or 20 days of hTERT induction; HT1080-LT shown as positive control for enhanced telomere length; mean telomeric signal (FITC) plotted bar graph (left). [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 ) TRF2 occupancy ChIP-qPCR on the IL1R1 promoter spanning +200 to –1000 bp of TSS in HT1080 cells following 0, 1, 6, or 12 days of hTERT induction in ex vivo culture; primers and normalisation as in ; IL1R1-3’UTR or a region 20 kb upstream used as negative controls. [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( J ) IL1R1 mRNA expression (normalised to GAPDH ) in HT1080 cells following 0, 1, 6, or 12 days of hTERT induction in ex vivo culture. [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( K ) TRF2 occupancy by ChIP-qPCR on the IL1R1 promoter in MDAMB23 or MDAMD231-LT cells using primers and normalisation described earlier. [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( L ) Expression for IL1R1 in MDAMB231 cells or MDAMD231-LT cells; GAPDH was used for normalisation. [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—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values.

Article Snippet: Antibody , TRF2 (mouse monoclonal) , Millipore , (Millipore 4A794)- , Flow cytometry (1:500).

Techniques: ChIP-qPCR, Ex Vivo, Binding Assay, Expressing, Flow Cytometry, Immunofluorescence, Microscopy, Staining, Positive 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 ) IL1R1 expression by qRT PCR following expression of TRF2-flag or TRF2 silencing (48 hr of transient transfection); GAPDH was used for normalisation. [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( B–C ) TRF2 ChIP-qPCR spanning +200 to –1000 bp of IL1R1 TSS for TRF2 occupancy ( B ) and promoter activity (luciferase reporter including –1500 bp TSS); ( C ) following expression of flag-tag TRF2 full-length, or the mutants TRF2-delB or TRF2-delM; anti-flag antibody used for ChIP; IL1R1-3’UTR or a region 20 kb upstream were used as negative controls. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( D ) TRF2 and IL1R1 levels by western blots following TRF2 induction with 2 or 4 µg/ml doxycycline (Dox) in HT1080 cells (stably transformed for Dox-inducible TRF2); GAPDH as loading. ( E ) TRF2 and IL1R1 expression in control or TRF2-induced HT1080 cells by flow-cytometry; TRF2/IL1R1 in x-axis in log scale for 20,000 cells. ( F ) IL1R1 levels in control or TRF2-induced HT1080 cells. CD44 used for cell-surface marker and nuclei were stained with DAPI; 25 cells in each condition were scored and plotted in the summary graph. [N=25] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( G ) TRF2 (left panel), IL1R1 (right panel) levels from xenograft tumours 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 graphs. [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 2—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 2—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 2—source data 3. Original image files for western blot or .

Article Snippet: Antibody , TRF2 (mouse monoclonal) , Millipore , (Millipore 4A794)- , Flow cytometry (1:500).

Techniques: Expressing, Quantitative RT-PCR, Transfection, ChIP-qPCR, Activity Assay, Luciferase, FLAG-tag, Western Blot, Stable Transfection, Transformation Assay, Control, Flow Cytometry, Marker, Staining, Fluorescence

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 ) IL1R1 protein levels on TRF2-flag induction (left) or TRF2 silencing (right) in HT1080 cells following 48 hr of transfection. ( B ) IL1R1 mRNA expression in MDAMB231 cells following expression of TRF2 cDNA or TRF2 silencing for 48 hr; GAPDH used as normalizing control (left). [N=3] Statistical significance was calculated using unpaired T test with Welch’s correction (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). IL1R1 protein levels in MDAMB231 cells following TRF2 induction for 48 hr by western blot (centre) and immuno-flow cytometry (right). ( C ) IL1R1 mRNA expression in HEK293T and MRC5 cells following transfection of TRF2-cDNA for 48 hr; GAPDH was used as normalizing control. [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 2—figure supplement 1—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 2—figure supplement 1—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 2—figure supplement 1—source data 3. Original image files for western blot for .

Article Snippet: Antibody , TRF2 (mouse monoclonal) , Millipore , (Millipore 4A794)- , Flow cytometry (1:500).

Techniques: Transfection, Expressing, Control, Western Blot, Flow Cytometry

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 ) Schematic depicting G4 motifs ( A, B ) and their respective positions on the IL1R1 promoter; sequence scheme depicting generic G4 motifs; ( B ) Circular dichroism profile (220–310 nm wavelength) of oligonucleotides (5 µM) with sequence of the G4 motifs A or B (or respective mutants with base substitutions) in solution. ( C ) ChIP-qPCR following BG4 ChIP at the IL1R1 promoter (fold enrichment over mock) overlaid with TRF2 occupancy (fold enrichment over IgG) in HT1080 cells. [N=3] Statistical significance was calculated using Šídák’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( D ) IL1R1 promoter activity in HT1080 cells from luciferase-reporter without or with G4-deforming substitutions against G4 motif A (IL1R1G4-MUT-A) or G4 motif B (IL1R1-G4 MUT-B) in presence or absence of TRF2 induction; G4 motifs A and B as shown in schematic; specific base substitutions have been indicated in blue font. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( E ) IL1R1 promoter activity, mRNA and protein levels in presence/absence of the G4 binding ligand 360 A with or without TRF2 induction. For Left and centre panels- [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( F ) IL1R1 promoter-firefly luciferase reporter cassette, with or without substitutions deforming the G4 motif B, was artificially inserted at the CCR5 locus using CRISPR-cas9 gene editing in HEK293T cells (scheme in left panel). Promoter activity from (right panel). [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( G ) TRF2 occupancy by ChIP-qPCR (right panel) at the artificially inserted IL1R1 promoter without or with G4-deforming substitutions (IL1R1G4 MUT-B);ChIP-qPCR primers designed specific to the inserted promoter using the homology arms(see Materials and methods). [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 3—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 3—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 3—source data 3. Original image files for western blot for .

Article Snippet: Antibody , TRF2 (mouse monoclonal) , Millipore , (Millipore 4A794)- , Flow cytometry (1:500).

Techniques: Sequencing, Circular Dichroism, ChIP-qPCR, Activity Assay, Luciferase, Binding Assay, CRISPR, 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 ) TRF2 ChIP-seq peak with sequence of the G4 motifs ( A, B ) and their respective positions on the IL1R1 promoter. ( B ) Occupancy of TRF2 by ChIP-qPCR at the IL1R1 promoter at the region of 0–200 bp of TSS in presence of ligand 360 A in three independent experiments in HT1080 cells. The dosage of 360 A used has been previous standardised [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( C, D ) Base substitutions for the G4 motif B in the IL1R1 promoter (as shown in ) were introduced using CRSIPR in HEK293T cells. TRF2 ChIP-qPCR spanning the IL1R1 promoter (left) and IL1R1 mRNA expression (right) in HEK293T cells with or without the G4-disrupting substitution in two replicates. [N=2]. Error bars correspond to SEM from independent experiments. Figure 3—figure supplement 1—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values.

Article Snippet: Antibody , TRF2 (mouse monoclonal) , Millipore , (Millipore 4A794)- , Flow cytometry (1:500).

Techniques: ChIP-sequencing, Sequencing, ChIP-qPCR, Expressing

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 ) Enrichment of histone marks H3K27ac, H3K4me3, H3K27me3, H3K9me3 (normalised to total H3) by ChIP-qPCR on the IL1R1 promoter in HT1080 cells in TRF2-up (induced) condition or un-induced corresponding control cells. [N=3] Statistical significance was calculated using Šídák’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( B ) Transcription factors/histone remodelers on the IL1R1 proximal promoter (–750 bp) across seven cell lines curated from ENCODE was plotted using UCSC genome browser hg19 human genome assembly. P300 enrichment marked in red box. ( C ) Immunoprecipitation using TRF2 antibody probed for p300, acp300/CBP, TRF2 or TRF1 (positive control) in input, TRF2 IP fraction or mock IP fraction in HT1080 cells. ( D ) Histone acetyl transferase (HAT) assay using purified histone H3/H4 and full-length p300 as substrate in presence/absence of recombinant TRF2; BSA (bovine serum albumin) was used as a non-specific mock protein. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). Error bars correspond to SEM from independent experiments. Figure 4—figure supplement 1—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 4—figure supplement 1—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 4—figure supplement 1—source data 3. Original image files for western blot for .

Article Snippet: Antibody , TRF2 (mouse monoclonal) , Millipore , (Millipore 4A794)- , Flow cytometry (1:500).

Techniques: ChIP-qPCR, Control, Immunoprecipitation, Positive Control, HAT Assay, Purification, Recombinant, 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 ) H3K27ac occupancy at the IL1R1 promoter spanning +200 to –1000 bp of TSS by ChIP-qPCR in HT1080 (left panel) and MDAMB231 cells (right panel) in control (uninduced), TRF2-induced (up) or TRF2-down conditions; IL1R1-3’UTR or a region 20 kb upstream were used as negative controls. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( B ) p300 occupancy on the IL1R1 promoter in HT1080 (left panel) and MDAMB231 cells (right panel) in control (uninduced), TRF2-induced or TRF2-down conditions; IL1R1-3’UTR or a region 20 kb upstream were used as negative controls. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( C ) CBP (left panel) and ac-p300/CBP (right panel) occupancy on the IL1R1 promoter in HT1080 cells in control (uninduced) or TRF2-induced conditions; IL1R1-3’UTR or a region 20 kb upstream were used as negative controls. [N=3] Statistical significance was calculated using Šídák’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( D ) p300, CBP, acp300/CBP and H3K27Ac occupancy on the IL1R1 promoter in HT1080 and HT1080-LT cells; IL1R1-3’UTR or a region 20 kb upstream were used as negative controls. [N=3] Statistical significance was calculated using Šídák’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). All protein ChIP, other than histones, were normalised to 1% input and fold-change have been calculated over respective IgG. Histone ChIP were normalised to 1% Input and fold change over total H3 have been calculated for individual samples (Further details in the Materials and methods section). Error bars correspond to SEM from independent experiments. Figure 4—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values.

Article Snippet: Antibody , TRF2 (mouse monoclonal) , Millipore , (Millipore 4A794)- , Flow cytometry (1:500).

Techniques: ChIP-qPCR, 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 ) Schematic showing positions (amino acid residues) where acetylation and deacetylation of TRF2 have been reported. ( B ) IL1R1 mRNA expression post 48 hr of transient over-expression of various TRF2 acetylation mutants. GAPDH was used for normalisation. [N=3] Statistical significance was calculated using Dunnett’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( C ) IL1R1 protein level following post 48 hr of transient over-expression of various TRF2 acetylation mutants; GAPDH used as loading control. ( D ) Immunofluorescence for IL1R1 in HT1080 cells without (control) or with induction of flag-tag TRF2 WT or mutant TRF2-293R; quantification from 29 cells in each case shown in graph. [N=29] Statistical significance was calculated using Mann-Whitney’s non-parametric test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( E ) Occupancy of flag-tag TRF2-WT or TRF2-293R on the IL1R1 promoter by ChIP-qPCR in HT1080 cells (left panel); IL1R1-3’UTR or a region 20 kb upstream were used as negative controls. Occupancy by ChIP-qPCR of p300 (middle panel) and acp300/CBP (right panel) without (control) or with induction of TRF2-WT and TRF2-293R. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( F ) TRF2-wild type (WT) and TRF2-2293R mutant proteins fused with dCAS9 expressed and targeted to the IL1R1 promoter using IL1R1 -specific gRNA in HT1080 and MDAMB231 cells. Following this, expression of IL1R1 or other TRF2 target genes (non-specific with respect to the IL1R1-gRNA) in HT1080 (left) or MDMB231 cells (right). [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). Error bars correspond to SEM from independent experiments. Figure 5—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 5—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 5—source data 3. Original image files for western blot for .

Article Snippet: Antibody , TRF2 (mouse monoclonal) , Millipore , (Millipore 4A794)- , Flow cytometry (1:500).

Techniques: Expressing, Over Expression, Control, Immunofluorescence, FLAG-tag, Mutagenesis, ChIP-qPCR, 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 ) TRF2-WT Flag or TRF2 K293R-Flag was stably over expressed in HT1080 cells and immunoprecipitation was performed using Anti-Flag antibody or Mock IgG. Enrichment for p300 in IP faction was tested for TRF2-WT (left) and TRF2 K293R(right). ( B ) Previously reported TRF2 target genes were checked following transient over-expression of TRF2 WT and TRF2 K293R in HT1080 cells. While K293R did not affect TRF2 mediated repression, it affected TRF2 activated genes PDGFR-B and IL1R1 but not WRNIP1. [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). Error bars correspond to SEM from independent experiments. Figure 5—figure supplement 1—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 5—figure supplement 1—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 5—figure supplement 1—source data 3. Original image files for western blot for .

Article Snippet: Antibody , TRF2 (mouse monoclonal) , Millipore , (Millipore 4A794)- , Flow cytometry (1:500).

Techniques: Stable Transfection, Immunoprecipitation, Over Expression, 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, 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: Antibody , TRF2 (mouse monoclonal) , Millipore , (Millipore 4A794)- , Flow cytometry (1:500).

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 ) TRF2-wild type (WT) and TRF2-2293R mutant proteins fused with dCAS9 expressed and targeted to the IL1R1 promoter using IL1R1-specific gRNA in HT1080 (left) or MDAMB231 cells (right). Following this, NFKB and p-NFKB (Ser536-phosphorylation) levels were assessed by flow-cytometry in ten thousand HT1080 or MDAM231 cells; mean fluorescence signal for IL1R1 and ratio of p-NFKB/total NFKB plotted in graphs on right panels. [N=1]. ( B ) IL1R1 knockout (KO) HT1080 cells using CRISPR were made and were transduced (lentiviral) to a doxycycline-inducible-TRF2 stable line (top left). TRF2, NFKappa-B and Ser536p-NFKappa-B in IL1R1 knockout or control HT1080 cells with or without TRF2 induction by doxycycline; GAPDH used as loading control (top right). Induction of TRF2 in HT1080 WT and IL1R1 KO cells to check for NFkappaB downstream target gene expression (bottom). Activation of NFKB target genes (~two- to fivefold) post-TRF2 over-expression was lower in IL1R1KO cells compared to WT cells (~10tenfold) [N=3] Statistical significance was calculated using Tukey’s multiple comparisons test (p values: *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ( C ) Phospho-NF-Kappa-B-(Ser-536p) and NFkappaB in HT1080 or HT1080-LT cells with or without IL1B stimulation (top panel); ratio of p-NFKB to total NFKB from above western blots plotted from two independent experiments (bottom panel). The NFkappa B (p65/RELA) is detected just below the 70 Kda molecular weight marker. Error bars correspond to SEM from independent experiments. Figure 6—figure supplement 1—source data 1. Source data for all plots in and corresponding details of statistical tests and p-values. Figure 6—figure supplement 1—source data 2. PDF file containing original western blot for indicating the relevant bands and treatments. Figure 6—figure supplement 1—source data 3. Original image files for western blot for .

Article Snippet: Antibody , TRF2 (mouse monoclonal) , Millipore , (Millipore 4A794)- , Flow cytometry (1:500).

Techniques: Mutagenesis, Phospho-proteomics, Flow Cytometry, Fluorescence, Knock-Out, CRISPR, Control, Targeted Gene Expression, Activation Assay, Over Expression, Western Blot, Molecular Weight, Marker

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: Scheme depicting relatively low infiltration of TAM in tumours with relatively long telomeres vis-à-vis tumours with short telomeres. Reduced non-telomeric TRF2 binding at the IL1R1 promoter in tumours with long telomeres, and consequent low IL1R1 activation, attenuated p65-mediated IL1-beta and macrophage infiltration. Created with BioRender.com .

Article Snippet: Antibody , TRF2 (mouse monoclonal) , Millipore , (Millipore 4A794)- , Flow cytometry (1:500).

Techniques: Binding Assay, Activation Assay

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: Antibody , TRF2 (mouse monoclonal) , Millipore , (Millipore 4A794)- , Flow cytometry (1:500).

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

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: Antibody , TRF2 (mouse monoclonal) , Millipore , (Millipore 4A794)- , Flow cytometry (1:500).

Techniques: Expressing

Journal: iScience

Article Title: Human SKI component SKIV2L regulates telomeric DNA-RNA hybrids and prevents telomere fragility

doi: 10.1016/j.isci.2024.111096

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-TRF2 (Clone 4A794) , Millipore , Cat#05-521; RRID: AB_2303145.

Techniques: Virus, Recombinant, Protease Inhibitor, Reverse Transcription, Blocking Assay, Mass Spectrometry, SYBR Green Assay, Flow Cytometry, Imaging, Mutagenesis, Cell Cycle Assay, shRNA, Software