Journal: Oncoimmunology

Article Title: Association of TRF2 expression and myeloid-derived suppressor cells infiltration with clinical outcome of patients with cutaneous melanoma

doi: 10.1080/2162402X.2021.1901446

Figure Lengend Snippet: Representative images of immune biomarkers and TRF2 staining, and their cell detection mask overlays used in the digital image analysis. Original magnification, x 200

Article Snippet: Formalin-fixed paraffin-embedded (FFPE) serial 4 μm tissue sections were freshly cut, deparaffinized, pre-treated, and stained with monoclonal antibodies (Abs) directed against CD33 (clone SP266, ready-to-use, Roche, Tucson, AZ, USA), CD14 (clone EP128, dilution 1/200, Epitomics, Burlingame, CA, USA), CD15 (clone MMA, ready-to-use, Roche, Tucson, AZ, USA), and TRF2 (clone 4A794.15, dilution 1/500, OriGene, Rockville, MA, USA) on a BenchMark ULTRA autostainer (Ventana Medical Systems, Tucson, AZ, USA).

Techniques: Staining

Journal: Oncoimmunology

Article Title: Association of TRF2 expression and myeloid-derived suppressor cells infiltration with clinical outcome of patients with cutaneous melanoma

doi: 10.1080/2162402X.2021.1901446

Figure Lengend Snippet: Correlative analysis between the clinical and histomolecular characteristics of the patients and the analyzed biomarkers in the metastatic melanoma cohort. *χ2-test, Student’s t-test or ANOVA test were used to investigate difference between groups.

Article Snippet: Formalin-fixed paraffin-embedded (FFPE) serial 4 μm tissue sections were freshly cut, deparaffinized, pre-treated, and stained with monoclonal antibodies (Abs) directed against CD33 (clone SP266, ready-to-use, Roche, Tucson, AZ, USA), CD14 (clone EP128, dilution 1/200, Epitomics, Burlingame, CA, USA), CD15 (clone MMA, ready-to-use, Roche, Tucson, AZ, USA), and TRF2 (clone 4A794.15, dilution 1/500, OriGene, Rockville, MA, USA) on a BenchMark ULTRA autostainer (Ventana Medical Systems, Tucson, AZ, USA).

Techniques: Biomarker Discovery, Mutagenesis

Journal: Oncoimmunology

Article Title: Association of TRF2 expression and myeloid-derived suppressor cells infiltration with clinical outcome of patients with cutaneous melanoma

doi: 10.1080/2162402X.2021.1901446

Figure Lengend Snippet: Multivariate analysis for overall survival in the cohort population

Article Snippet: Formalin-fixed paraffin-embedded (FFPE) serial 4 μm tissue sections were freshly cut, deparaffinized, pre-treated, and stained with monoclonal antibodies (Abs) directed against CD33 (clone SP266, ready-to-use, Roche, Tucson, AZ, USA), CD14 (clone EP128, dilution 1/200, Epitomics, Burlingame, CA, USA), CD15 (clone MMA, ready-to-use, Roche, Tucson, AZ, USA), and TRF2 (clone 4A794.15, dilution 1/500, OriGene, Rockville, MA, USA) on a BenchMark ULTRA autostainer (Ventana Medical Systems, Tucson, AZ, USA).

Techniques: Biomarker Discovery

Journal: bioRxiv

Article Title: Telomere-Dependent Interleukin-1 Receptor Activation Promotes Immune Suppression in Triple-Negative-Breast Cancer

doi: 10.1101/2021.12.07.471419

Figure Lengend Snippet: Mapped reads on the IL1R1 promoter for TRF2 ChIP-seq (Mukherjee et al, 2019) in HT1080 cells (replicate 1 and 2) and respective inputs for normalization; plotted using publicly available software IGV (Integrated Genomics viewer) with sequence alignment files.

Article Snippet: TRF2 shRNA was procured from Origene ( TL308880 ).

Techniques: ChIP-sequencing, Software, Sequencing

Journal: bioRxiv

Article Title: Telomere-Dependent Interleukin-1 Receptor Activation Promotes Immune Suppression in Triple-Negative-Breast Cancer

doi: 10.1101/2021.12.07.471419

Figure Lengend Snippet: A. Occupancy of TRF2 at the IL1R1 promoter was checked in xenograft tumors (N=5) and HT1080 cells by chromatin immunoprecipitation (ChIP) followed by qPCR. Primers spanning +200 bp from TSS (transcription start site) to -1000 bp were used; regions 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. B. Occupancy of TRF2 at the IL1R1 promoter was lower in xenograft tumors and HT1080-LT compared to HT1080 cells by ChIP followed by qPCR. C. IL1R1 protein levels in HT1080 and HT1080-LT cells by western blot; GAPDH was used as loading control. D. IL1R1 protein by immuno-flow cytometry in HT1080 and HT1080-LT cells with three independent replicates for each. IL1R1 expression was plotted along the X-axis in log scale. E. Expression of IL1R1 on cell surface by immunofluorescence microscopy in in HT1080 and HT1080-LT cells. Cells were stained with cell-mask red for visualization and DAPI for marking the cell nucleus. Average IL1R1 expression from 25 individual cells were plotted in the adjoining graph. F. Secreted IL1B (pg/ml) in supernatant media from HT1080 and HT1080-LT cells in two independent experiments. G. Relative telomere length by flow-cytometry based estimation of telomeric signal in HT1080 cells following 0, 1, 6, 12 or 20 days of TERT induction (Methods). HT1080-LT was used as a positive control for enhanced telomere length in the assay. H. TRF2 occupancy on the IL1R1 promoter in HT1080 cells following 0, 1, 6 or 12 days of TERT induction (see methods) in ex-vivo culture using primers and normalizations as stated earlier in . I. IL1R1 mRNA expression (normalized to GAPDH ) in HT1080 cells following 0, 1, 6 and 12 days of TERT induction (see methods) in ex-vivo culture. J. TRF2 occupancy on the IL1R1 promoter in MDAMB231 cells and MDAMD231-LT (long telomere) cells using primers and normalizations as stated earlier in . K. Expression for IL1R1 and other relevant cytokines: IL1A,IL1B, IL2,IL6,IL8 and TNF in MDAMB231 cells and MDAMD231-LT cells. GAPDH was used for normalization. Statistical significance has been calculated using Mann-Whitney’s non-parametric test for individual graphs where N≥5. In case of independent repetitions of the same experiment, unpaired T test with Welsh’s correction was performed for significance (p values : * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001). Error bar correspond to standard error of mean from three independent experiments unless N is stated otherwise.

Article Snippet: TRF2 shRNA was procured from Origene ( TL308880 ).

Techniques: Chromatin Immunoprecipitation, Negative Control, Binding Assay, Western Blot, Control, Flow Cytometry, Expressing, Immunofluorescence, Microscopy, Staining, Positive Control, Ex Vivo

Journal: bioRxiv

Article Title: Telomere-Dependent Interleukin-1 Receptor Activation Promotes Immune Suppression in Triple-Negative-Breast Cancer

doi: 10.1101/2021.12.07.471419

Figure Lengend Snippet: TRF2 occupancy at telomeres following TRF2 ChIP in xenograft tumors (C) and ex-vivo cells in HT1080 or HT1080-LT cells (D).

Article Snippet: TRF2 shRNA was procured from Origene ( TL308880 ).

Techniques: Ex Vivo

Journal: bioRxiv

Article Title: Telomere-Dependent Interleukin-1 Receptor Activation Promotes Immune Suppression in Triple-Negative-Breast Cancer

doi: 10.1101/2021.12.07.471419

Figure Lengend Snippet: TRF2 occupancy at telomeres following TRF2 ChIP in TERT-inducible HT1080 cells at different number of days of induction.

Article Snippet: TRF2 shRNA was procured from Origene ( TL308880 ).

Techniques:

Journal: bioRxiv

Article Title: Telomere-Dependent Interleukin-1 Receptor Activation Promotes Immune Suppression in Triple-Negative-Breast Cancer

doi: 10.1101/2021.12.07.471419

Figure Lengend Snippet: Relative telomere length by qRT-PCR (H); TRF2 occupancy at telomeres following TRF2 ChIP (I); and, secreted IL1B (ng/ml) (J) in MDAMB 231 or MDAMD231-LT cells in two independent experiments. Statistical significance using t-Test with Welch’s correction (p values : * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001)

Article Snippet: TRF2 shRNA was procured from Origene ( TL308880 ).

Techniques: Quantitative RT-PCR

Journal: bioRxiv

Article Title: Telomere-Dependent Interleukin-1 Receptor Activation Promotes Immune Suppression in Triple-Negative-Breast Cancer

doi: 10.1101/2021.12.07.471419

Figure Lengend Snippet: A. IL1R1 expression was checked by qRT PCR post over-expression of TRF2-flag construct or TRF2 silencing by siRNA (see methods) transiently for 48 hrs. in HT1080 cells. B. IL1R1 protein expression was checked following over-expression of TRF2-flag construct for 48 hrs in HT1080 cells. GAPDH was used as a loading control in the experiments. C. TRF2 was induced in stable-TRF2-inducible-HT1080 cells (see methods) by treatment with 2 µg/ml or 4 µg/ml doxycycline in media for 48 hrs. TRF2 induction was checked by western blot and subsequently IL1R1 expression was checked by western blot. GAPDH was used as a loading control in the experiments. D. IL1R1 levels on the cell surface was checked in control and TRF2 over-expressing HT1080 cells. CD44 was used as a cell surface marker and nuclei were stained with DAPI; 25 individual cells were scored for IL1R1 expression in control and TRF2 over-expressing cells and plotted in the summary graph. E. IL1R1 in control and TRF2 induced HT1080 cells by flow-cytometry. TRF2 and IL1R1 was plotted along the x-axis in log scale for 20 thousand cells. F. IL1R1 promoter activity (see methods) in control (vector only) or transient TRF2-flag-expression for 48 hrs in HT1080 cells. G. IL1R1 promoter activity (see methods) in control (scrambled) or following TRF2-siRNA transfection for 48 hrs in HT1080 cells. H. TRF2 WT (flagged tag) or mutants delB and delM were transiently transfected in HT1080 cells and cells harvested after 48hrs were used for ChIP using antibody for flag-tag. Subsequently occupancy was checked on IL1R1 promoter by qRT PCR using primers as previously stated. I. IL1R1 promoter activity was checked (see methods) in control (vector only) along with TRF2-flag, delB and delM over-expressed conditions for 48 hrs in HT1080 cells. J. Circular dichroism profile (220-310 nm wavelength) of oligonucleotides (5 µM) bearing sequence of the G4 motifs (or base substituted mutants) present in the IL1R1 promoter (within 200 bp upstream of TSS) in solution. K. IL1R1 promoter occupancy (fold enrichment over mock) was checked by qRT PCR following BG4 ChIP and overlaid with TRF2 occupancy (fold enrichment over IgG) in HT1080 cells. L. IL1R1 promoter activity was checked in control or on TRF2 over-expression (48 hrs) for the IL1R1 WT luciferase construct or the G4 motif mutants A and B in HT1080 cells. M-O. IL1R1 promoter activity, mRNA and protein expression in control and TRF2 over-expressed condition (48 hrs post transfection) in presence or absence of the G4 binding ligand 360A (2 µM). P-R. IL1R1 promoter (with our without the G4 motif mutant B) along with downstream firefly luciferase was inserted at the CCR5 locus by CRISPR-cas9 gene editing in HEK293T cells (see methods). Firefly signal (promoter activity) was checked in IL1R1 WT or the G4 mutant inserts followed by TRF2 ChIP at the inserted promoter; TRF2 ChIP at telomeres was used as positive control. Statistical significance has been calculated using Mann-Whitney’s non-parametric test for individual graphs where N≥5. In case of independent repetitions of the same experiment, unpaired T test with Welsh’s correction was performed for significance (p values : * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001). Error bar correspond to standard error of mean from three independent experiments unless N is stated otherwise.

Article Snippet: TRF2 shRNA was procured from Origene ( TL308880 ).

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Construct, Control, Western Blot, Marker, Staining, Flow Cytometry, Activity Assay, Plasmid Preparation, Transfection, FLAG-tag, Circular Dichroism, Sequencing, Luciferase, Binding Assay, Mutagenesis, CRISPR, Positive Control

Journal: bioRxiv

Article Title: Telomere-Dependent Interleukin-1 Receptor Activation Promotes Immune Suppression in Triple-Negative-Breast Cancer

doi: 10.1101/2021.12.07.471419

Figure Lengend Snippet: A-C. TRF2 knockdown in HT1080 cells using TRF2-specific shRNA (A) and previously reported siRNA (B) following 48 hrs of transfection. Over-expression of TRF2 WT (flag-tag) and DNA-binding mutants, delB and delM in HT1080 cells following 48 hrs of transfection (C). D. TRF2 knockdown in MDAMB231 cells using TRF2 specific shRNA post 48 hrs of transfection. E. IL1R1 mRNA expression in MDAMB231 cells post transfection of TRF2 WT cDNA (over-expression) and TRF2 shRNA (silencing) for 48 hrs. GAPDH was used as a normalizing control. F-G. IL1R1 protein expression in MDAMB231 cells post TRF2 over-expression for 48 hrs by western blot (F) and immuno-flow cytometry (G). H. TRF2 occupancy on the IL1R1 promoter in HT1080, MDAMB231, HEK293T and MRC5 immortalized cells by Chromatin immunoprecipitation (ChIP) followed by qPCR. Primers spanning +200 to -1000 bp of TSS were used to check 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. I. IL1R1 mRNA expression in HEK293T and MRC5 immortalized cells post transfection of TRF2 WT cDNA (over-expression) for 48 hrs. GAPDH was used as normalizing control. J-K. G4 motifs (A and B) shown on the IL1R1 promoter upstream of TSS and G4 disrupting mutations marked on the motifs. L. IL1R1 mRNA expression (normalized to GAPDH ) in HT1080 cells following treatment with different concentrations of the G4 binding ligand 360A. For assessment of statistical significance of independent repetitions of the same experiment, unpaired T test with Welsh’s correction was performed for significance (p values : * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001). Error bar correspond to standard error of mean from three independent experiments unless N is stated otherwise.

Article Snippet: TRF2 shRNA was procured from Origene ( TL308880 ).

Techniques: Knockdown, shRNA, Transfection, Over Expression, FLAG-tag, Binding Assay, Expressing, Control, Western Blot, Flow Cytometry, Chromatin Immunoprecipitation, Negative Control

Journal: bioRxiv

Article Title: Telomere-Dependent Interleukin-1 Receptor Activation Promotes Immune Suppression in Triple-Negative-Breast Cancer

doi: 10.1101/2021.12.07.471419

Figure Lengend Snippet: A. Enrichment of histone marks H3K27ac, H3K4me3, H3K27me3, H3K9me3 (normalized to total H3) on the IL1R1 promoter in HT1080 cells in TRF2 high (induced) condition and corresponding control cells. B. ENCODE curated transcription factors/histone remodelers on the IL1R1 proximal promoter (up to 1500 bp from TSS) across seven cell lines was plotted using UCSC genome browser hg19 human genome assembly. P300 marked in red box for the two sites of enrichment. C. TRF2 WT or DNA binding mutants, delB or delM were transiently over-expressed in HT1080 cells in TRF2-silenced background; expression of TRF2 and respective mutants (probed by anti-flag antibody) and p300. GAPDH as used as loading control. D. p300 levels in control and TRF2 induced condition following cycloheximide treatment at 0, 12 and 24 hrs. E. p300, IL1R1 and TRF2 protein levels in HT1080 or HT1080-LT cells; GAPDH used as loading control. F. Immunofluorescence microscopy for p300 in HT1080 or HT1080-LT cells. Mean p300 signal for 25 individual cells plotted in graph below. G-I. TRF2 PTM mutants (K176R, K179R, K190R, K293R) and TRF2 WT transiently expressed and confirmed by TRF2 western (G); mRNA expression (normalized to GAPDH ) (H) and protein levels for IL1R1 (I) in HT1080 cells after 48 hrs; GAPDH used as loading control in the western blots. Statistical significance has been calculated using Mann-Whitney’s non-parametric test for individual graphs where N≥5. For assessment of statistical significance of independent repetitions of the same experiment, unpaired T test with Welsh’s correction was performed for significance (p values : * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001). Error bar correspond to standard error of mean from three independent experiments unless N is stated otherwise.

Article Snippet: TRF2 shRNA was procured from Origene ( TL308880 ).

Techniques: Control, Binding Assay, Expressing, Immunofluorescence, Microscopy, Western Blot

Journal: bioRxiv

Article Title: Telomere-Dependent Interleukin-1 Receptor Activation Promotes Immune Suppression in Triple-Negative-Breast Cancer

doi: 10.1101/2021.12.07.471419

Figure Lengend Snippet: A. H3K27ac occupancy (normalized to total H3) over 1% input was checked on the IL1R1 promoter in HT1080 and MDAMB231 cells in TRF2 high (cDNA) or low (shRNA) conditions. B. p300 occupancy on the IL1R1 promoter in HT1080 and MDAMB231 cells in TRF2 high (cDNA) and low (shRNA) conditions. C-D. CBP and ac-p300/CBP occupancy on the IL1R1 promoter in HT1080 cells in control or TRF2-induced conditions. E. Immunoprecipitation (IP) in HT1080 cells using TRF2 antibody probed for p300, acp300/CBP, TRF2 or TRF1 (positive control) in input, TRF2 IP fraction or mock IP fraction. F. Occupancy on the IL1R1 promoter in HT1080 and HT1080-LT cells for p300, CBP and acp300/CBP. H3K27ac occupancy (normalized to total H3) over 1% input on the IL1R1 promoter in the respective cell lines. G. IL1R1 protein by immunofluorescence in HT1080 cells following transient over-expression of TRF2 WT-flag and the mutant TRF2-293R-flag (48 hrs post transfection). F. acp300/CBP, p300 and flag-tag occupancy on the IL1R1 promoter in HT1080 cells in control and cells transiently over-expressing TRF2 WT-flag or the mutant TRF2-293R-flag (48 hrs post transfection). I. Histone acetyl transferase (HAT) assay was performed (see methods) using histone H3/H4 as a substrate for full length p300 in presence or absence of purified mammalian TRF2. All protein ChIPs, other than histones, were normalized to 1% input and fold-change shown over respective IgGs. Statistical significance has been calculated using Mann-Whitney’s non-parametric test for individual graphs where N≥5. In case of independent repetitions of the same experiment, unpaired T test with Welsh’s correction was performed for significance (p values : * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001). Error bar correspond to standard error of mean from three independent experiments unless N is stated otherwise

Article Snippet: TRF2 shRNA was procured from Origene ( TL308880 ).

Techniques: shRNA, Control, Immunoprecipitation, Positive Control, Immunofluorescence, Over Expression, Mutagenesis, Transfection, FLAG-tag, Expressing, HAT Assay, Purification

Journal: bioRxiv

Article Title: Telomere-Dependent Interleukin-1 Receptor Activation Promotes Immune Suppression in Triple-Negative-Breast Cancer

doi: 10.1101/2021.12.07.471419

Figure Lengend Snippet: A. Scheme showing the IL1 pathway (cognate receptors and ligands) that leads to NFKappaB (p65/RELA) activation via sub-cellular intermediates. B-D. IL1B treatment (10 ng/ml) in HT1080 and MDAMB231 cells led to higher NFKappaB activation in cells with high TRF2. Activation of NFKappaB signaling was confirmed through Ser536 phosphorylation of NFKappaB (normalized to total NFKappaB) up to 12 hours following IL1B stimulation in HT1080 (B) and MDAMB231 (C) cells; further, phospho-inhibitory-Kappa-B (p-IKB in (B)) and NFkappaB activation in HT1080 versus HT1080-LT cells following IL1B stimulation (D) was consistent with TRF2-dependent activation of IL1R1 . E. NFKappaB downstream targets –IL6, IL8 and TNF were checked in HT1080 cells post IL1A, IL1B and TNF A treatment (10 ng/ml respectively) for 24 hrs in control (scrambled siRNA) or TRF2 low (TRF2 siRNA mediated knockdown) conditions respectively. F. NFKappaB downstream targets –IL6, IL8 and TNF were checked in HT1080 cells post IL1B treatment (10 ng/ml) for 24 hrs in control or TRF2 over-expressed conditions in presence/absence of IL1 receptor antagonist (IL1RA; 20 ng/ml). In case of independent repetitions of the same experiment, unpaired T test with Welsh’s correction was performed for significance (p values : * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001). Error bar correspond to standard error of mean from three independent experiments unless N is stated otherwise.

Article Snippet: TRF2 shRNA was procured from Origene ( TL308880 ).

Techniques: Activation Assay, Phospho-proteomics, Control, Knockdown

Journal: bioRxiv

Article Title: Telomere-Dependent Interleukin-1 Receptor Activation Promotes Immune Suppression in Triple-Negative-Breast Cancer

doi: 10.1101/2021.12.07.471419

Figure Lengend Snippet: A. HT1080 cells transiently transfected with TRF2 siRNA or scrambled control siRNA and treated with IL1A, IL1B or TNF-alpha (10 ng/ml respectively) after 48 hrs of transfection. IL1R1, TRF2, p65-Ser536 phosphorylation, total p65 and GAPDH (as loading control) probed by western blot following 3 hours of cytokine treatment. B. p65 downstream targets: IL6, IL8 and TNF in HT1080 cells following treatment with IL1RA treatment (5,10 or 20 ng/ml) for 24 hrs. GAPDH was used as for normalization. C. Secreted IL1B (pg/ml) in control or stable TRF2-expressing HT1080 cells in two independent replicates.

Article Snippet: TRF2 shRNA was procured from Origene ( TL308880 ).

Techniques: Transfection, Control, Phospho-proteomics, Western Blot, Expressing

Journal: bioRxiv

Article Title: Telomere-Dependent Interleukin-1 Receptor Activation Promotes Immune Suppression in Triple-Negative-Breast Cancer

doi: 10.1101/2021.12.07.471419

Figure Lengend Snippet: A-B. TRF2 and IL1R1 levels in xenograft tumors in NOD SCID mice (see methods; control and TRF2 induced HT1080 cells; N=6) was assessed by immuno-flow cytometry and fluorescence signal for TRF2 and IL1R1 was plotted in the x-axis in log scale. Mean fluorescence signal from individual tumors in control and TRF2 induced tumors have been plotted in adjoining graphs. C. Percentage of TAM was estimated in xenograft tumors (HT1080 control and HT1080-TRF2 induced) using markers CD11b and CD206. The percentage values HT1080 control and HT1080-TRF2 induced tumors were plotted in the adjoining summary graph. D. mRNA expression for key cytokines- IL1A,IL1B, IL2,IL6,IL8 , TNF, IL10,IL4 and IL13 were checked in xenograft tumors (HT1080 control and HT1080- TRF2 induced); 18S was used for normalization. E. Scheme depicting relatively low infiltration of TAM in tumors with longer telomeres vis-à-vis tumors with short telomeres. Reduced non-telomeric TRF2 binding at the IL1R1 promoter in tumors with long telomeres, and consequent low IL1R1 activation, attenuated p65-mediated IL1-beta and macrophage infiltration. Figure created with BioRender. Com. F. Heat map summarizing mRNA expression of key cytokines across the model systems used in the study: TNBC tissue, TNBC derived organoids, xenograft tumors and ex-vivo cells in relation to telomere length. Fold change of gene expression (long/short telomeres) color coded as per the reference legend along y-axis. Statistical significance has been calculated using Mann-Whitney’s non-parametric test for individual graphs (p values : * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001)

Article Snippet: TRF2 shRNA was procured from Origene ( TL308880 ).

Techniques: Control, Flow Cytometry, Fluorescence, Expressing, Binding Assay, Activation Assay, Derivative Assay, Ex Vivo, Gene Expression

Journal: bioRxiv

Article Title: Telomere-Dependent Interleukin-1 Receptor Activation Promotes Immune Suppression in Triple-Negative-Breast Cancer

doi: 10.1101/2021.12.07.471419

Figure Lengend Snippet: A. mRNA expression of IL1R1 in xenograft tumors (HT1080 control or HT1080-TRF2 induced); human 18S was used for normalization. B. mRNA expression of mouse M2 macrophage markers CD163 and CD206 in xenograft tumors (HT1080 control or HT1080-TRF2 induced); human 18S was used for normalization. C. Invasion assay in HT1080 control or TRF2-induced cells in presence/absence of THP1-derived M2 macrophages. Invaded HT1080 cells were stained for crystal violet stain for visualization. Statistical significance has been calculated using Mann-Whitney’s non-parametric test for individual graphs (p values : * ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, **** ≤ 0.0001)

Article Snippet: TRF2 shRNA was procured from Origene ( TL308880 ).

Techniques: Expressing, Control, Invasion Assay, Derivative Assay, Staining

Journal: Nucleic acids research

Article Title: PRL-3 promotes telomere deprotection and chromosomal instability.

doi: 10.1093/nar/gkx392

Figure Lengend Snippet: Figure 2. RAP1 and TRF2-dependent recruitment of PRL-3 to telomere. (A) In situ PLA analysis of PRL-3’s associations with RAP1 and TRF2. HCT116 cells were pre-extracted, fixed, inmunostained with indicated pairs of antibodies and probed with Duolink in situ PLA reagent. Binding foci were in red and dashed lines indicated outline of nucleus (determined by DAPI counter staining). Scale bar, 10 m. (B) TRF2- and RAP1-dependent recruitment of PRL-3 to telomeric DNA in vitro. Purified myc-TRF2 (150 ng), His-RAP1 (120 ng), and His-PRL-3 (30 ng) were co-incubated with 1 g biotin-labeled telomere (lanes 1–4) or Alu (lanes 5–8) probe as indicated and subjected to pull-down analysis with Streptavidin agarose. Precipitates were analyzed by western blot with antibodies to TRF2, RAP1 and PRL-3. (C and D) TRF2 and RAP1-dependent recruitment of PRL-3 to telomere in cells. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h, pre-extracted, fixed and subjected to IF-FISH staining. (C) Representative PRL-3 association with telomere. Scale bar, 10 m. Areas in white squares were enlarged. (D) Quantification of cells with ≥5 associations between PRL-3 foci and telomere. Mean ± SD of three independent experiments. n > 100 cells per single experiment. Student’s t-test. (E) Knockdown efficiencies of RAP1 and TRF2. HCT116 cells were transfected with 50 nM siRNAs against RAP1 or TRF2 for 48 h. Lysates were analyzed by western blot with indicated antibodies. (F) ChIP analysis of PRL-3 binding to telomeric and Alu DNA. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h and processed for ChIP using anti-PRL-3 or pre-immune IgG. Upper, representative blots of hybridization with probe to telomere or Alu. Input, 2% DNA. Lower, quantification of relative optical densities (OD). Relative OD was calculated by normalizing to OD of Input and relative OD of control siRNA-transfected sample was set as 100%. Mean ± SD of three independent experiments. Student’s t-test.

Article Snippet: For transient knockdown of PRL-3, following siRNAs (synthesized by GenePharma, Shanghai, China) were used: PRL-3 #1, sense: 5′-ACAAACACAUGCGCUUCCUdT dT-3′, antisense: 5′-AGGAAGCGCAUGUGUUUGUdT dT-3′; PRL-3 #2, sense: 5′-UUGAGGACCUGAAGAAG UAdTdT-3′, antisense: 5′-UACUUCUUCAGGUCCUC AAdTdT-3′; PRL-3 #3, sense: 5′-CAGCUCCUGUGUG GAGAAAdTdT-3′, antisense: 5′-UUUCUCCACACAG GAGCUGdTdT-3′; PRL-3 #4, sense: 5′-GACCAGAUG CUCAUGUGUUdTdT-3′, antisense: 5′-AACACAUGA GCAUCUGGUCdTdT-3′; control, sense: 5′-UUUUCCG AACGUGUCACGUdTdT-3′, antisense: 5′-ACGUGAC ACGUUCGGAAAAdTdT-3′. siRNA pools specific for RAP1 (SR 310061) and TRF2 (SR304782) were obtained from OriGene. siRNAs (50 nM) were transfected into cells cultured in 60 mm plates with Lipofectamine 2000 reagent.

Techniques: In Situ, Binding Assay, Staining, In Vitro, Purification, Incubation, Labeling, Western Blot, Transfection, Knockdown, Hybridization, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Telomere shortening and metabolic compromise underlie dystrophic cardiomyopathy

doi: 10.1073/pnas.1615340113

Figure Lengend Snippet: Cardiomyocytes of DMD mouse model exhibit telomere dysfunction. (A) Schematic of telomeric ends protected by shelterin proteins. (B) Endogenous expression levels of telomere capping genes were determined by RT-qPCR in primary cardiomyocytes: Trf1, Trf2, Tpp1, Tin2, Pot1a, and Pot1b (n = 4–5 mice per genotype; technical replicates n = 2). (C) Percentage of DNA damage response marker 53bp1 positivity was quantified by flow cytometry relative to IgG control (gray). (D) 53bp1-positive cardiomyocytes (percentage total) are shown (n = 3 mice per genotype; n = 5,000 cardiac troponin T–positive cells per sample). All assays were performed using 8-wk-old animals. Data are represented as mean ± SEM. Statistical analyses entailed one-way ANOVA with post hoc Bonferroni correction. *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: Terf2 , Mm01253557_m1 , Life Tech.

Techniques: Expressing, Quantitative RT-PCR, Marker, Flow Cytometry, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Telomere shortening and metabolic compromise underlie dystrophic cardiomyopathy

doi: 10.1073/pnas.1615340113

Figure Lengend Snippet: Taqman probes used in this study

Article Snippet: Terf2 , Mm01253557_m1 , Life Tech.

Techniques:

Journal: Cell reports

Article Title: Persistent NF-κB activation in muscle stem cells induces proliferation-independent telomere shortening

doi: 10.1016/j.celrep.2021.109098

Figure Lengend Snippet:

Article Snippet: Primers purchased from Applied Biosystems and included IKBKB-FAM (Gene assay ID: Mm01222247_m1), CHUK-FAM (Mm00432529_m1), IKBKG-FAM (Mm00494927_m1), XRCC5-FAM (Mm00550142_m1), XRCC6-FAM (Mm00487458_m1), TERF2-FAM (Mm01253555_m1), TERF1-FAM (Mm00436928_m1), TERF2IP-FAM (Mm01243676_m1), TPP1-FAM (Mm00487016_m1), TINF2-FAM (Mm00461166_g1), Pot1B-FAM (Mm01278790_m1), Tert-FAM (Mm00436931_m1), and GAPDH-VIC (4352339e).

Techniques: Recombinant, Electron Microscopy, Plasmid Preparation, Avidin-Biotin Assay, Blocking Assay, Saline, Lysis, Imaging, Kinase Assay, Staining, Generated, Software, Microscopy

Journal: Nucleic Acids Research

Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

doi: 10.1093/nar/gkx392

Figure Lengend Snippet: RAP1 mediates PRL-3–TRF2 interaction. ( A ) Precipitation of endogenous RAP1 and TRF2 by GST-PRL-3. HCT116 cell lysates (500 μg) were co-incubated with 1 μg purified GST (lane 2) or GST-PRL-3 (lane 3), and subjected to GST pull-down with Glutathione-agarose beads. Precipitates and 50 μg HCT116 cell lysates (lane 1, input) were analyzed by western blot with indicated anti-shelterin antibodies. Purities of GST-PRL-3 and GST were verified by Coomassie blue staining (lower panel). ( B ) Endogenous PRL-3 associates with RAP1 and TRF2 in cells in a DNA/RNA-independent manner. HCT116 cell lysates (500 μg) were immunoprecipitated by 1 μg antibody against PRL-3 (upper panel) or RAP1 (lower panel). For control, 1 μg preimmune IgG was used. Parts of lysates were also pre-treated with benzonase (Benz) for 30 min at room temperature before immunoprecipitation. Precipitates and 25 μg HCT116 cell lysates (input) were subjected to western blot. ( C ) Requirement of RAP1 for PRL-3-TRF2 association in vitro . Purified proteins (100 ng each) were mixed as indicated (lanes 4–11) and subjected to GST pull-down assay. Some of mixtures were also pre-treated with benzonase for 30 min at room temperature (lanes 8–11). Precipitates and purified proteins (10 ng each, lanes 1–3, input) were analyzed by western blot with antibodies to TRF2, RAP1 and PRL-3. ( D ) Enhancement of PRL-3–TRF2 interaction by RAP1 in cells. COS7 cells were co-transfected with indicated amounts of pcDNA3 and pcDNA3-myc-RAP1 plasmids. The total amount of plasmids for each sample was adjusted to 2 μg. After 48 h, cells were harvested and lysates were immunoprecipitated with anti-PRL-3 and analyzed by western blot with antibodies to TRF2, myc-tag, RAP1 and PRL-3. ( E ) Requirement of RAP1 for PRL-3–TRF2 association in cells. HCT116 cells were transfected with 50 nM control or RAP1-specific siRNA for 48 h. Cell lysates were immunoprecipitated with anti-PRL-3. HC, IgG heavy chain. ( F ) Upper, GST pull-down assay to map the domain of RAP1 required for its interaction with PRL-3. A total of 100 ng GST (lane 2) or GST-RAP1s (lanes 3–8) was co-incubated with 100 ng His-PRL-3 (lanes 2–8). After pull-down, precipitates were detected by anti-PRL3 and anti-GST. Input, 10 ng His-PRL-3 (lane 1). Lower, summary of binding. FL, full-length RAP1; ΔB, deletion of BRCT domain; ΔBΔM, deletion of BRCT and Myb domains; ΔCΔRΔN, deletion of coiled-coil, RCT and NLS domains. Red asterisks, position of GST or GST fusion proteins. ( G ) Adaptor function of RAP1 in mediating TRF2 and PRL-3 interaction is dependent on its Myb and RCT domains. Purified FALG-TRF2, GST-RAP1 (FL, ΔBΔM, ΔCΔRΔN) and His-PRL-3 proteins (100 ng each) were mixed as indicated. Five percent of mixtures were kept as input, and the rests were subjected to pull-down with anti-FLAG-agarose bead. Precipitates and input were analyzed by western blot with antibodies to PRL-3, TRF2 and GST-tag. ( H ) Blockade of PRL-3's recruitment to RAP1–TRF2 complex by GFP-Myb. HCT116 cells were transfected with 0.5 μg of pEGFP-N1 or pEGFP-N1-Myb plasmid for 48 h. Lysates (500 μg) were immunoprecipitated with 1 μg anti-RAP1 or pre-immune IgG. Precipitates and 25 μg lysates (input) were analyzed by western blot.

Article Snippet: Recombinant FLAG-TRF2, myc-TRF2 and myc-PRL-3 (all from OriGene) were expressed in human HEK293 cells and purified.

Techniques: Incubation, Purification, Western Blot, Staining, Immunoprecipitation, In Vitro, Pull Down Assay, Transfection, Binding Assay, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

doi: 10.1093/nar/gkx392

Figure Lengend Snippet: RAP1 and TRF2-dependent recruitment of PRL-3 to telomere. ( A ) In situ PLA analysis of PRL-3's associations with RAP1 and TRF2. HCT116 cells were pre-extracted, fixed, inmunostained with indicated pairs of antibodies and probed with Duolink in situ PLA reagent. Binding foci were in red and dashed lines indicated outline of nucleus (determined by DAPI counter staining). Scale bar, 10 μm. ( B ) TRF2- and RAP1-dependent recruitment of PRL-3 to telomeric DNA in vitro . Purified myc-TRF2 (150 ng), His-RAP1 (120 ng), and His-PRL-3 (30 ng) were co-incubated with 1 μg biotin-labeled telomere (lanes 1–4) or Alu (lanes 5–8) probe as indicated and subjected to pull-down analysis with Streptavidin agarose. Precipitates were analyzed by western blot with antibodies to TRF2, RAP1 and PRL-3. ( C and D ) TRF2 and RAP1-dependent recruitment of PRL-3 to telomere in cells. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h, pre-extracted, fixed and subjected to IF-FISH staining. (C) Representative PRL-3 association with telomere. Scale bar, 10 μm. Areas in white squares were enlarged. (D) Quantification of cells with ≥5 associations between PRL-3 foci and telomere. Mean ± SD of three independent experiments. n > 100 cells per single experiment. Student's t -test. ( E ) Knockdown efficiencies of RAP1 and TRF2. HCT116 cells were transfected with 50 nM siRNAs against RAP1 or TRF2 for 48 h. Lysates were analyzed by western blot with indicated antibodies. ( F ) ChIP analysis of PRL-3 binding to telomeric and Alu DNA. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h and processed for ChIP using anti-PRL-3 or pre-immune IgG. Upper, representative blots of hybridization with probe to telomere or Alu. Input, 2% DNA. Lower, quantification of relative optical densities (OD). Relative OD was calculated by normalizing to OD of Input and relative OD of control siRNA-transfected sample was set as 100%. Mean ± SD of three independent experiments. Student's t -test.

Article Snippet: Recombinant FLAG-TRF2, myc-TRF2 and myc-PRL-3 (all from OriGene) were expressed in human HEK293 cells and purified.

Techniques: In Situ, Binding Assay, Staining, In Vitro, Purification, Incubation, Labeling, Western Blot, Transfection, Hybridization

Journal: Nucleic Acids Research

Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

doi: 10.1093/nar/gkx392

Figure Lengend Snippet: Silencing of PRL-3 promotes DDR and senescence. ( A ) Efficiencies of PRL-3 silencing in HCT116 (knockdown by two shRNAs using lentivirus system, left) and SW480 (knockout by CRISPR/Cas9 system, right) cells and its effects on indicated protein levels. WT, wild-type. KO, knockout. ( B ) Effects of PRL-3 silencing on phosphorylations of H2AX and CHK1. Samples treated with 20 μM etoposide (ETP) for 4 h were used as positive controls. ( C ) Effects of PRL-3 silencing on TIF formation. Indicated HCT116 cells were subjected to IF-FISH staining. Upper, representative staining. Arrows, colocalizations between γH2AX and telomere (TIFs). Scale bar, 5 μm. Lower, quantification of cells with ≥5 TIF. Mean ± SD of two independent experiments. n > 200 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 silencing on anaphase bridges (APB) and micronuclei (MN) formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. n > 1000 cells scored per sample for MN and n > 50 anaphase cells scored per sample for APB. Student's t -test. Representative images of APB (red arrow) and MN (white arrow) of HCT116 cells stained with DAPI were shown. ( E ) ChIP analysis of RAP1 and TRF2's binding to telomeric or Alu DNA in HCT116 and S480 cells silenced for PRL-3. Upper, representative blots after ChIP with indicated antibodies or IgG. Input, 2% DNA. Lower, quantification of relative OD. Relative OD was calculated by normalizing to that of input and relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( F ) PRL-3 silencing induced ROS-dependent cellular senescence and DNA damage response. Indicated HCT116 cells were treated with NAC (10 mM), GSH (10 mM) or DMSO (1:1000) for 24 h. Part of cells were fixed and processed for β-galactosidase staining, others were analyzed by western blot. Upper, representative β-galactosidase staining of cells treated with DMSO. Middle, quantification of β-galactosidase positive cells. Mean ± SD of two independent experiments. n > 400 cells per single experiment. Student's t -test. Lower, western blot of γH2AX.

Article Snippet: Recombinant FLAG-TRF2, myc-TRF2 and myc-PRL-3 (all from OriGene) were expressed in human HEK293 cells and purified.

Techniques: Knock-Out, CRISPR, Staining, Binding Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

doi: 10.1093/nar/gkx392

Figure Lengend Snippet: Overexpression of PRL-3 promotes telomere dysfunction. ( A ) Validation of PRL-3 stable overexpression. WI38 fibroblasts were infected with control or PRL-3-expressing letivirus. Expression vectors pcDNA3-myc-PRL-3 (for HCT116 cells), pcDNA3.1-myc-PRL-3 (for LoVo cells) and the respective control plasmids were transfected into cells, followed by selection and pooling of stable colonies. Cell lysates were examined by western blot with antibodies to PRL-3, TRF2 and RAP1. ( B ) Effects of PRL-3 stable overexpression on γH2AX, pCHK1 and p53 levels. Indicated cells were treated with ETP (20 μM) or DMSO (1:1000) for 4 h. ( C ) Effects of PRL-3 stable overexpression on TIF formation. WI38 cells were analyzed by IF-FISH staining of pATM (green) and telomere (red). Left, representative staining. Arrows, foci of TIFs. Scale bar, 5 μm. Right, quantification of cells with ≥5 TIFs. Mean ± SD of two independent experiments. n > 60 metaphase per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on dysfunctional telomere repair pathways. Upper, representative CO-FISH staining of WI38 cells. Metaphase cells were stained with probes specific for leading (red) and lagging (green) strands and counterstained with DAPI (blue). Yellow arrow, a typical T-SCE. White arrow, a chromosome–chromosome fusion. Red arrowhead, a MTS. Scale bar, 2.5 μm. Lower, quantification of abnormalities. Mean ± SD of two independent experiments. n > 1300 chromosomes per single experiment. Student's t -test. ( E ) Southern blot analysis of PRL-3 stable overexpression-induced telomere deprotection. Genomic DNA from indicated cells were resolved on agarose gel, transferred to nitrocellulose membrane and probed with biotin-labeled telomere probe. ( F ) qPCR analysis of PRL-3 stable overexpression-induced telomere deprotection. Relative telomere to single copy gene (T/S) ratio of control cells was set as 1. Mean ± SD of three independent experiments. n = 4 replicates per single experiment. Student's t -test.

Article Snippet: Recombinant FLAG-TRF2, myc-TRF2 and myc-PRL-3 (all from OriGene) were expressed in human HEK293 cells and purified.

Techniques: Over Expression, Infection, Expressing, Transfection, Selection, Western Blot, Staining, Southern Blot, Agarose Gel Electrophoresis, Labeling

Journal: Nucleic Acids Research

Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

doi: 10.1093/nar/gkx392

Figure Lengend Snippet: PRL-3 relocates RAP1 and TRF2 from telomeric DNA. ( A ) Effects of PRL-3 stable overexpression on the chromatin abundance of RAP1, TRF2 and TRF1. Nuclei from HCT116 cells were homogenized in buffer containing indicated concentrations of NaCl. Chromatin-enriched fractions were analyzed by western blot. Left, representative blots. Right, relative levels of TRF2, RAP1 and TRF1. Protein band were scanned and relative OD was calculated by normalizing to OD of H2B. The relative OD of sample prepared with 150 mM NaCl was set as 100%. Mean ± SD of three independent experiments. ANOVA. ( B ) Effects of PRL-3 stable overexpression on bindings of RAP1 and TRF2 to telomeric and Alu DNA. Indicated cells were crosslinked, immunoprecipitated with antibodies to RAP1, TRF2 or pre-immune IgG, and precipitated DNA was analyzed by ChIP. Upper, representative blots. Lower, quantification of relative OD, which was calculated by normalizing to that of Input. Relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on telomere associations of RAP1 and TRF2 in WI38 cells. Left, representative IF-FISH staining of telomere (red) and RAP1 or TRF2 (green). Arrows, foci of co-localization. Scale bar, 10 μm. Right, quantification of cells with ≥5 associations between RAP1 or TRF2 foci and telomere. Mean ± SD of two independent experiments. n > 80 cells per single experiment. Student's t -test. ( D ) EMSA analysis of PRL-3, RAP1 and TRF2's associations with telomeric DNA. Indicated concentrations of purified FLAG-TRF2, His-RAP1, myc-PRL-3 were co-incubated with Biotin-labeled telomere probe (20 nM). To induce super-shift, 0.1 μg anti-PRL-3 (lane 5), anti-TRF2 (lanes 6 and 18) and IgG (lane 7) were used. Note that anti-PRL-3 and anti-TRF2-induced super-shifts of Complex II partially co-migrated with Complex I (lanes 5 and 6).

Article Snippet: Recombinant FLAG-TRF2, myc-TRF2 and myc-PRL-3 (all from OriGene) were expressed in human HEK293 cells and purified.

Techniques: Over Expression, Western Blot, Immunoprecipitation, Staining, Purification, Incubation, Labeling

Journal: Nucleic Acids Research

Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

doi: 10.1093/nar/gkx392

Figure Lengend Snippet: Disrupting PRL-3-RAP1 complex or expressing ectopic TRF2 attenuates PRL-3 overexpression-promoted telomere deprotection, DNA damage, chromosomal instability and senescence. ( A ) HCT116 control and PRL-3 overexpressing cells were transfected with 0.5 μg of pEGFP-N1-Myb or pEGFP-N1 plasmid for 72 h, and indicated proteins were analyzed by western blot. ( B ) qPCR analysis of telomere length of cells in (A). T/S ratio of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test. ( C ) Quantification of micronuclei of cells in (A). Mean ± SD of two independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Quantification of β-galactosidase-positive cells in (A). Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( E ) Relative migration of cells in (A). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of two independent experiments. Three replicates per single experiment. Student's t -test. ( F ) HCT116 control and PRL-3 overexpressing cells were infected with control (Lv-con) or TRF2-expressing lentivirus (Lv-TRF2) for 120 h, and lysates were subjected to western blot. ( G ) qPCR analysis of telomere length of cells in (F). T/S ratio of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. 3 replicates per single experiment. Student's t -test. ( H ) Quantification of micronuclei of cells in (F). Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( I ) Quantification of β-galactosidase-positive cells in (F). Mean ± SD of three independent experiments. n > 300 cells per single experiment. Student's t -test. ( J ) Relative migration of cells of (F). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test.

Article Snippet: Recombinant FLAG-TRF2, myc-TRF2 and myc-PRL-3 (all from OriGene) were expressed in human HEK293 cells and purified.

Techniques: Expressing, Over Expression, Transfection, Plasmid Preparation, Western Blot, Migration, Infection