mouse monoclonal anti-trf2 clone 4a794  (Millipore)

Journal: iScience

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

doi: 10.1016/j.isci.2024.111096

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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

Journal: Nucleic Acids Research

Article Title: TRF2 positively regulates SULF2 expression increasing VEGF-A release and activity in tumor microenvironment

doi: 10.1093/nar/gkz041

Figure Lengend Snippet: TRF2 regulates the amount of VEGF-A in the secretome of tumor cells. ( A ) Luminex/XMAP multiplexed analysis of CMs derived from TRF2-compromised (shTRF2) or -overexpressing (pBabe-TRF2) HCT116 cells and their control counterparts (shScramble/pBabe). CMs were collected 48 h after cell starvation and the expression levels of a panel of secreted chemokines and growth factors involved in angiogenesis were quantified. For each analyte, results are expressed as log 2 fold change of protein levels in silenced/overexpressing cells over their controls. ( B ) Detailed analysis of VEGF-A concentration from A. Histograms show the mean values (±SD) of a single experiment performed in triplicate (* P < 0.1; ** P < 0.01; *** P < 0.001; Student's t -test). ( C ) Concentration of VEGF-A was evaluated by ELISA in the CM of HCT116 silenced (shTRF2_N1 and shTRF2_N2) or overexpressing (pBabe-TRF2) TRF2 , collected 48 h after serum-starvation. As control, the amount of VEGF-A was assayed in the CM of HCT116 cells not infected (parental) or infected with viral particles delivering control vectors (shSCR or pBabe). Results were normalized to cell number. Histograms show the mean (±SD) of at least three independent experiments performed in triplicate (* P < 0.1, ** P < 0.01, *** P < 0.001; Student's t -test).

Article Snippet: Immunostaining was performed using the mouse monoclonal antibodies anti-TRF2 (clone 4A794; Millipore, Billerica, MA, USA), and anti SULF2 (clone G4, Santa Cruz Biotechnology) in an automated stainer (Bond Max III, Leica Biosystem, Milan, Italy) according to the manufacturer's instructions.

Techniques: Luminex, Derivative Assay, Control, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Infection

Journal: Nucleic Acids Research

Article Title: TRF2 positively regulates SULF2 expression increasing VEGF-A release and activity in tumor microenvironment

doi: 10.1093/nar/gkz041

Figure Lengend Snippet: TRF2 modulation affects the secretome of tumor cells and the angiogenic response of endothelial cells. ( A ) Tubule formation assay. HUVEC cells were seeded on Matrigel ® in the presence of the CMs deriving from HCT116 silenced (shTRF2_N1 and shTRF2_N2) or overexpressing (pBabe-TRF2) TRF2 . CMs from cells transduced with control vectors (shScramble or pBabe) or uninfected (parental) were used as controls. EBM-2 supplemented or not with VEGF-A (100 ng/ml) was used as positive and negative control, respectively. The CMs were collected at the indicated times (12, 24 and 48 h). Histograms show the mean number of branching points calculated on five different fields and expressed as fold induction over the negative control. Pictures in the lower panels show representative images of tubular-like structures (5X magnification). Graphs show the mean ±SD of at least three independent experiments performed in triplicate (* P < 0.1, ** P < 0.01, *** P < 0.001; Student's t -test). (B, C) In vivo evaluation of angiogenic response induced by CMs from HCT116 cells silenced (shTRF2_N2) ( B ) or overexpressing (pBabe-TRF2) TRF2 ( C ). For each experiment the CMs from control cells (shScramble or pBabe) were also assayed. EBM-2 and EBM-2 supplemented with VEGF-A (100 ng/ml) and TNFα (2 ng/ml) were used as negative and positive controls, respectively. Where indicated, CM was incubated with an anti-VEGF-A 165 blocking antibody (1 μg/ml for 30 minutes) and assayed. Left panels : representative pictures show Matrigel ® plugs recovered 5 days post-injection. Right panels : histograms represent the average hemoglobin content (±SD) measured in the relative samples and expressed as absorbance (OD 540 nm )/mg of Matrigel ® ( n = 8 plugs per group; * P < 0.1, ** P < 0.01, *** P < 0.001; Student's t -test).

Article Snippet: Immunostaining was performed using the mouse monoclonal antibodies anti-TRF2 (clone 4A794; Millipore, Billerica, MA, USA), and anti SULF2 (clone G4, Santa Cruz Biotechnology) in an automated stainer (Bond Max III, Leica Biosystem, Milan, Italy) according to the manufacturer's instructions.

Techniques: Tube Formation Assay, Transduction, Control, Negative Control, In Vivo, Incubation, Blocking Assay, Injection

Journal: Nucleic Acids Research

Article Title: TRF2 positively regulates SULF2 expression increasing VEGF-A release and activity in tumor microenvironment

doi: 10.1093/nar/gkz041

Figure Lengend Snippet: TRF2 affects the binding of secreted VEGF-A 165 to membrane HSPGs. ( A ) Amount of membrane-associated VEGF-A 165 was quantified by ELISA. The analysis was performed on membrane-enriched lysate fractions obtained from HCT116 cells silenced (shTRF2) or overexpressing (pBabe-TRF2) TRF2 and their controls (shScramble and pBabe). Results were normalized to the total amount of membrane proteins. ( B ) Immunofluorescence analysis of membrane associated VEGF-A on viable HCT116 cells silenced for TRF2 (shTRF2) and their control (shSCR) counterpart. Left panel : fluorescence-activated cell sorting (FACS) analysis. Histogram shows the fluorescence intensities in the negative control (black line) and in HCT116 silenced (shTRF2, red line) or not (shSCR, blue line) for TRF2. Right panel , results of confocal-microscopy. Representative images acquired at 63x magnification and their respective 3D surface plots. ( C ) Concentration of VEGF-A evaluated by ELISA in the CM obtained from HCT116 overexpressing (pBabe-TRF2) or not (pBabe) TRF2 , untreated or treated with CoCl 2 (100 μM for 16 h), heparin (200 ng/ml for 16 h) or heparinase II (15 mU/ml for 2 h). For CM, cells were growth for 24 h in serum-free medium and all the stimuli were directly added in medium. All the histograms show the mean ±SD of at least three independent experiments (* P < 0.1, ** P < 0.01, *** P < 0.001; Student's t -test).

Article Snippet: Immunostaining was performed using the mouse monoclonal antibodies anti-TRF2 (clone 4A794; Millipore, Billerica, MA, USA), and anti SULF2 (clone G4, Santa Cruz Biotechnology) in an automated stainer (Bond Max III, Leica Biosystem, Milan, Italy) according to the manufacturer's instructions.

Techniques: Binding Assay, Membrane, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Control, Fluorescence, FACS, Negative Control, Confocal Microscopy, Concentration Assay

Journal: Nucleic Acids Research

Article Title: TRF2 positively regulates SULF2 expression increasing VEGF-A release and activity in tumor microenvironment

doi: 10.1093/nar/gkz041

Figure Lengend Snippet: Extracellular release of VEGF-A is regulated by TRF2 through the control of SULF2 expression. ( A ) Gene expression of SULF2 was evaluated by qPCR in HCT116 cells silenced (shTRF2) or overexpressing (pBabe-TRF2) TRF2 and in their control counterparts (shScramble and pBabe). Results are expressed as fold change of mRNA levels in silenced/overexpressing cells over their controls, after β-actin normalization. ( B ) IF analysis of SULF2 expression in cells silenced or overexpressing TRF2 and their controls. Representative images acquired at 63x magnification are shown. ( C ) Expression of TRF2 ( left panel ) and SULF2 ( right panel ) was evaluated by qPCR in HCT116 cells overexpressing the wild-type (pBabe TRF2) or the mutated forms of TRF2 (pBabe TRF2ΔM and pBabe TRF2ΔBΔM) and their control counterpart (pBabe). Results are expressed as fold change of mRNA levels in overexpressing cells over their controls, after β-actin normalization. ( D ) Schematic representation of putative TRF2-binding sites located upstream the transcription starting site (TSS) of SULF2 . ( E ) Real-time qPCR analysis of TRF2-chromatin immunoprecipitates. DNA regions containing (Chr.2 sub-telomeric region) or not (RPLP0) TTAGGG sequences were used as positive and negative control, respectively. ( F ) pGL3-promoter vector (pGL3 empty) and the vector containing the wild-type or mutant form of the distal regulatory element were co-transfected with a renilla vector (pRL-TK) in control (pBabe) and TRF2 silenced (shTRF2) HCT116 cells and luciferase activity was assayed. Firefly luciferase signal was normalized for the renilla signal to derive the relative luciferase activity. Results are expressed as fold change over the activity measured in the cells transfected with the control vector. The histograms show the mean ± SE of at least three independent experiments performed in triplicate (* P < 0.1, ** P < 0.01, *** P < 0.001; Student's t -test).

Article Snippet: Immunostaining was performed using the mouse monoclonal antibodies anti-TRF2 (clone 4A794; Millipore, Billerica, MA, USA), and anti SULF2 (clone G4, Santa Cruz Biotechnology) in an automated stainer (Bond Max III, Leica Biosystem, Milan, Italy) according to the manufacturer's instructions.

Techniques: Control, Expressing, Gene Expression, Binding Assay, Negative Control, Plasmid Preparation, Mutagenesis, Transfection, Luciferase, Activity Assay

Journal: Nucleic Acids Research

Article Title: TRF2 positively regulates SULF2 expression increasing VEGF-A release and activity in tumor microenvironment

doi: 10.1093/nar/gkz041

Figure Lengend Snippet: SULF2 is a direct target of TRF2 and controls tumor angiogenesis. ( A ) Concentration of VEGF-A evaluated by ELISA in the CM of empty vector or SULF2 overexpressing HCT116 cells infected with scramble (shSCR), TRF2 (shTRF2) or SULF2 (shSULF2) targeting shRNAs. Results were normalized to cell number. (B, C) CMs described in A were assayed for their capability of inducing capillary structures in HUVEC cells. ( B ) Mean number of branching points calculated on five different fields and expressed as fold induction over the negative control. ( C ) Representative images showing tubular-like structures (5X magnification). ( D ) Concentration of VEGF-A evaluated in the CM obtained from HCT116 cells infected as indicated. CM were obtained from cells growth in serum-free medium for 24 h. Results were normalized to cell number. (E, F) CMs described in D were assayed for their angiogenic potential in vitro . ( E ) Mean number of branching points calculated as in B. ( F ) representative tubular-like structure images from E. The histograms show the mean ± SD of at least three independent experiments performed in triplicate (* P < 0.1, ** P < 0.01, *** P < 0.001; Student's t -test).

Article Snippet: Immunostaining was performed using the mouse monoclonal antibodies anti-TRF2 (clone 4A794; Millipore, Billerica, MA, USA), and anti SULF2 (clone G4, Santa Cruz Biotechnology) in an automated stainer (Bond Max III, Leica Biosystem, Milan, Italy) according to the manufacturer's instructions.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Infection, Negative Control, In Vitro

Journal: Nucleic Acids Research

Article Title: TRF2 positively regulates SULF2 expression increasing VEGF-A release and activity in tumor microenvironment

doi: 10.1093/nar/gkz041

Figure Lengend Snippet: TRF2-silencing impairs tumor growth and metastasis by inhibiting SULF2-mediated angiogenesis. ( A ) Empty vector or SULF2 overexpressing (SULF2) HCT116 cells infected with lentiviral particles carriyng scramble (shSCR), TRF2 (shTRF2) or SULF2 (shSULF2) targeting shRNAs were intramuscularly injected in immunocompromised nude mice and tumor growth was assayed. Left panel : tumor weight was evaluated at the indicated days post-injection. The graph shows the mean ±SD from 5/7 mice per group (* P < 0.1, ** P < 0.01, *** P < 0.001; Student's t -test). Right panel: representative images showing the size of tumors excised at day 21 after cell injection. ( B ) IHC analyses of the tumors from A. Left panel : representative IHC images of tumor stained with antibodies against TRF2, SULF2 and CD31. Right panel : quantitative analysis of the indicated markers. Data are mean (±SD) from two mice per group (* P < 0.1, ** P < 0.01, *** P < 0.001; Mann–Whitney test). ( C ) Luminescent colon cancer cells were injected in the spleen of CB17-SCID mice and after 30 min the spleen was removed by spleenectomia. Real-time tumor dissemination was monitored by the IVIS imaging system 200 series (Caliper Life Sciences, Hopkinton, MA, USA) at day 14 and 21 after tumor cell injection. Left panel : Representative images of tumor dissemination acquired and analyzed using the Living Image Software version 3.0 (Caliper Life Sciences). Right panel : histogram reporting photons of tumor dissemination in each experimental group. The instrumental limit of photons detection for these cell lines was 1 × 10 5 .

Article Snippet: Immunostaining was performed using the mouse monoclonal antibodies anti-TRF2 (clone 4A794; Millipore, Billerica, MA, USA), and anti SULF2 (clone G4, Santa Cruz Biotechnology) in an automated stainer (Bond Max III, Leica Biosystem, Milan, Italy) according to the manufacturer's instructions.

Techniques: Plasmid Preparation, Infection, Injection, Staining, MANN-WHITNEY, Imaging, Software

Journal: Nucleic Acids Research

Article Title: TRF2 positively regulates SULF2 expression increasing VEGF-A release and activity in tumor microenvironment

doi: 10.1093/nar/gkz041

Figure Lengend Snippet: Direct correlation between TRF2 and SULF2 expression in colorectal cancer has prognostic impact on patient survival. (A, B) The correlation between TRF2 and SULF2 expression was evaluated by immunohistochemistry (IHC) using the immune-reactive score (IRS). ( A ) Box plots show the median values of SULF2 expression in the 56 TRF2 Low and in the 113 TRF2 High CRCs (* P = 0.046; Student's t -test). ( B ) Optimal SULF2 cut-off (IRS > 15) was established by ROC analysis and correlation with TRF2 was calculated. Pie charts show the distribution of SULF2 (low and high) in the sub-populations of TRF2 Low and TRF2 High CRC patients (** P = 0.008; χ 2 test). (C, D) Correlation between TRF2 expression and microvessel density (evaluated by CD31). ( C ) Box plots show average number of vessels evaluated in a sub-set of 30 TRF2 Low and 30 TRF2 High CRC patients (*** P = 0.0001). ( D ) Optimal CD31 cut-off (vessels/HPF >18) was established by ROC analysis and correlation with TRF2 was evaluated. The pie charts show the distribution of CD31 low and high in the 30 TRF2 Low and in the 30 TRF2 High CRCs (*** P = 0.0001; χ 2 test). ( E ) IHC evaluation of SULF2 and CD31 expression in two representative CRCs showing TRF2 Low and TRF2 High , respectively. Magnification 40x. Scale bar: 50 μm. ( F ) Box plot shows the SULF2 mRNA levels evaluated on a cohort of 327 TRF2 Low ( z score < 0) and 294 TRF2 High ( z score > 0) CRC patients from the TCGA dataset (* P = 0.01; Wilcoxon rank-sum test). ( G ) Disease-free survival (DFS) evaluated by Kaplan–Meier curves on CRC patients from the TCGA dataset. Patients were stratified on the basis of TRF2 and SULF2 mRNA expression and survival was evaluated in patient subgroups with positive TRF2 / SULF2 correlations ( TRF2 High / SULF2 High vs TRF2 Low / SULF2 Low ) (* P = 0.03; log-rank test).

Article Snippet: Immunostaining was performed using the mouse monoclonal antibodies anti-TRF2 (clone 4A794; Millipore, Billerica, MA, USA), and anti SULF2 (clone G4, Santa Cruz Biotechnology) in an automated stainer (Bond Max III, Leica Biosystem, Milan, Italy) according to the manufacturer's instructions.

Techniques: Expressing, Immunohistochemistry