Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Schematic representation of luciferase screening approach. Upper panel shows the four target predictions software used for in silico analysis. Bottom panel indicates the main steps performed in the high‐throughput screening. B Upper panel, sequence interaction of miR‐182‐3p with the target site of the wild type 3′UTR of TRF2 in human. Bottom panel, generation of mutant 3′UTR of TRF2 luciferase construct containing the deletion of target site for miR‐182‐3p. C–E Luciferase reporter assay in HeLa cells using the synthetic miR‐Control or miR‐182‐3p in combination with the wild type (C) or the mutant 3′UTR of TRF2 construct (D) or the wild type 3′UTR of TRF1 (E). F, G Western blotting for TRF2 expression in telomerase‐positive (HeLa, HCT116, MDA‐MB‐231, MDA‐MB‐436) and ALT‐positive (U2‐OS, Saos‐2) cells transiently transfected with miR‐Control or miR‐182‐3p. Upper panel shows the quantification of TRF2 expression. Bottom panel, representative images are shown, actin was used as loading control. H U2‐OS cells transiently transfected with the miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor were assayed by quantitative immunofluorescence for TRF2 3 days post‐transfection. Left panel, representative images. Scale bar: 10 μm. Right panel, quantification of TRF2 fluorescence intensity. a.f.u. arbitrary fluorescence units. N = number of analyzed nuclei. Red bar indicates mean value. I U2‐OS cells transfected as described in (H) were assayed by immunofluorescence combined with telomeric FISH. Left panel, representative images of co‐localizations between TRF2 and telomeres (white arrowheads). Scale bar: 10 μm. Right panel, co‐localizations were analyzed using ImageJ software. N = number of analyzed nuclei. Data information: For (C–G and I), data are shown as mean ± SD. Three independent experiments were performed ( n = 3). P values are determined by Student's t ‐test; for (H), P values are determined by Mann–Whitney t ‐test. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Luciferase, Software, In Silico, High Throughput Screening Assay, Sequencing, Mutagenesis, Construct, Reporter Assay, Western Blot, Expressing, Transfection, Immunofluorescence, Fluorescence, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Results of high‐throughput luciferase screening performed in Hela cells using the wild type 3′UTR‐TRF2 vector in combination with each of the 54 miRNAs selected by in silico analysis. Three days post‐transfection, luciferase ratio (Renilla:Firefly) of each miRNA was calculated, the control miRNA was set “1.” Renilla:Firefly ratios < 1 indicate target specificity of candidate miRNAs for the 3′UTR of TRF2. miRNAs near to the ratio of 0.5 were considered for further analysis. Two biological replicates were performed. B HeLa cells transiently transfected with the indicated miRNAs (miR‐Control, miR‐182‐3p, miR‐519e‐5p, miR‐296‐3p) were assayed by western blotting. Upper panel, quantification of TRF2 expression. Bottom panel, representative images of TRF2, TRF1 and RAP1 are shown, actin was used as loading control. C Analysis of TRF2 mRNA expression performed by qPCR in four different cancer cell lines (HeLa, MDA‐MB‐231, MDA‐MB‐436, U2‐OS) 3 days post‐transfection with miR‐Control or miR‐182‐3p. The control miRNA was set “1.” Three independent experiments were performed. D, E Telomeric ChIP assay in MDA‐MB‐231 (D) and U2‐OS cells (E). Quantification of TRF2 enrichment at telomeric repeats, in the different conditions, is shown in the table under the respective figure. Alu probe and Rabbit IgG were used as negative control for the assay. Data information: For (A), data are presented as mean values. For (B, C), data are presented as mean values ± SD and Student t‐ test was used to calculate statistical significance. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: High Throughput Screening Assay, Luciferase, Plasmid Preparation, In Silico, Transfection, Western Blot, Expressing, Negative Control

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A MDA‐MB‐231 cells were transiently transfected with the indicated miRNAs or siRNA. The indicated DNA damage markers were assayed by western blotting. Actin was used as loading control. B Telomeric DNA FISH performed in MDA‐MB‐231 transiently transfected with the indicated miRNAs. Telomere length was measured by TLF software and indicated as arbitrary fluorescence unit (a.f.u). N = number of analyzed nuclei. Black bar indicates mean value. C DNA damage markers were assayed by western blotting in HeLa cells. Actin was used as loading control. D Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in HeLa cells transfected with the indicated miRNAs or siRNAs. Co‐localizations of γH2AX with telomeres are indicated as mean number of TIFs per nucleus. E Representative images and enlargements of co‐localizations of experiment described in D. F Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in HeLa cells transfected with the indicated miRNAs or siRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. G Representative images of co‐localizations relative to the experiment described in (F). H, I MDA‐MB‐231 and HeLa cells over‐expressing TRF2 or an empty vector (pBabe) were transiently transfected with miR‐Control or miR‐182‐3p. TRF2, pATM and γH2AX expression were assayed by western blotting. Actin was used as loading control. Data information: For (D) and (F), data are presented as mean values ± SD. Three independent replicates were performed. Scale bar: 10 μm. At least 60 nuclei were analyzed in (D) and (F). A Student t‐ test was used to calculate statistical significance. For (B), P values are determined by Mann–Whitney t ‐test. All the experiments were performed 3 days post‐transfection with the indicated miRNAs or siRNAs. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Western Blot, Software, Fluorescence, Immunofluorescence, Expressing, Plasmid Preparation, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: Immunofluorescence analysis of γH2AX combined with telomeric FISH (TIFs) was performed in MDA‐MB‐231 cells transfected with the indicated miRNAs or siRNAs. The mean number of TIFs per nucleus was analyzed. Representative images and enlargements of co‐localizations (white arrowheads) relative to the experiment described in (A). Scale bar: 10 μm. Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in MDA‐MB‐231 cells transfected with the indicated miRNAs or siRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. Representative images of co‐localizations (white arrowheads) relative to the experiment described in (C). Scale bar: 10 μm. Quantification of TIFs in MDA‐MB‐231 cells over‐expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The mean number of TIFs per nucleus was quantified. Representative images and enlargements relative to the experiment described in (E). White arrowheads indicate co‐localizations events. Scale bar: 10 μm. Quantification of PIFs in MDA‐MB‐231 cells over‐expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. Representative images relative to the experiment described in (G). White arrowheads indicate co‐localizations events. Scale bar: 10 μm. Data information: For (A, C, E, G) data are shown as mean ± SD. Three independent experiments were performed ( n = 3). P values are determined by unpaired two‐tailed t‐ test. At least 60 nuclei were analyzed for each experimental condition. All the experiments were performed 3 days post‐transfection with the indicated miRNAs or siRNAs. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B MDA‐MB‐436 and MDA‐MB‐231 cells underwent two rounds of transfection with miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor. Starting from the day of the second transfection, cell confluence was monitored by Incucyte every 24 h up to a maximum of 3 days. The percentage of cell confluence was analyzed. C, D Cell number of MDA‐MB‐436 (C) and MDA‐MB‐231 (D) cells and TRF2 expression were analyzed by automatic cell count and by western blotting at the end of the experiment described in (A) and (B). Actin was used as loading control. E Two‐dimensional scatter plots of Annexin V analysis performed in MDA‐MB‐436 at the end of the second cycle of transfection with miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor. Red boxes indicate early and late apoptotic cells. F Quantification of Annexin V‐positive cells (%) of experiment described in (E). G Two‐dimensional scatter plots of Annexin V analysis performed in MDA‐MB‐231 as described in (E). H Quantification of Annexin V‐positive cells (%) of experiment described in (G). I, J MDA‐MB‐436 cells over‐expressing TRF2 or an empty vector (pBabe) were transiently transfected with indicated miRNAs and cell count (I) or apoptosis (J) analysis was performed 72 h post‐transfection. Data information: For (A, B) data are shown as mean ± SEM. For (C, D, F, H, I, J), data are shown as mean ± SD. For (A–D) and (I), three independent experiments were performed ( n = 3). P values are determined by unpaired two‐tailed t‐ test. For (F), (H) and (J), two different biological replicates were performed. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Expressing, Cell Counting, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Western blotting for TRF2 expression in BJ cells transiently transfected with miR‐Control or miR‐182‐3p. The graph represents the quantification of three independent experiments. Representative images are shown, Actin was used as loading control. Unspecific bands are indicated with (*). B, C Mean of γH2AX foci per nucleus was analyzed in BJ cells 72 h post‐transfection with the indicated miRNAs. Representative images of γH2AX foci are shown in (C). D Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in BJ cells 72 h post‐transfection with the indicated miRNAs. Left panel: The mean number of TIFs per nucleus was analyzed. Right panel: Representative images and enlargements of co‐localizations. E Cell number of BJ cells was analyzed by automatic cell count at the end of the second round of transfection with miR‐Control or miR‐182‐3p. F FACS analysis to evaluate cell cycle progression by Propidium Iodide (PI) staining in BJ cells treated as indicated in (E). G β‐Galactosidase assay in BJ cells after two rounds of transfection with mimic miR‐Control or miR‐182‐3p. Left panel: Analysis of β‐galactosidase‐positive cells. Right panel: Representative images. H–J IL‐6 (H), CXCL1 (I), IL‐8 (J) factors were analyzed by ELISA to evaluate the senescence‐associated secretory phenotype (SASP) in BJ cells treated as indicated in (G). Data information: For (A, B, D, E and G–J), a student t‐ test was used to calculate statistical significance. Scale bars (10 μm). P values are indicated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Western Blot, Expressing, Transfection, Immunofluorescence, Cell Counting, Staining, Enzyme-linked Immunosorbent Assay

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A TRF2 and γH2AX expression after two rounds of transfection with the indicated miRNAs, was analyzed by western blotting in MCF10A cells. Actin was used as loading control. B–E The mean number of γH2AX foci (B) and TIFs (D) per nucleus were analyzed 72 h post‐transfection with the indicated mimic miRNAs in MCF10A cells. Representative images (C) and (E) are referred to the experiment showed in (B) and (D) respectively. F, G Cell confluence (F) of MCF10A was monitored by Incucyte, every 24 h starting from the day of the second transfection, and cell number (G) was counted at the end of experiment (day 4). H–I Cell cycle progression analysis by PI staining (H) and cell death analysis by Annexin V assay (I) were performed in MCF10A upon two rounds of transfection with the indicated miRNAs. J β‐Galactosidase assay in MCF10A cells after two rounds of transfection with mimic miR‐Control or miR‐182‐3p. Left panel: Analysis of β‐galactosidase‐positive cells. Right panel: Representative images. Data information: Panels (B, D, F, G, J) data are presented as mean values ± SD. A Student t‐ test was used to calculate statistical significance. P values are indicated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, Transfection, Western Blot, Staining, Annexin V Assay

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B MDA‐MB‐231 (A) and MDA‐MB‐436 (B) tumor xenografts were treated with LNPs‐empty, LNPs‐miR‐Control or by LNPs‐miR‐182‐3p when the tumors became palpable. Mice were treated 6 times by intravenous tail vein injections with 20 μg of LNPs‐miR‐Control, LNPs‐miR‐182‐3p or equivalent volume of LNPs‐empty as indicated in the scheduling. The mean of tumor volumes ( n = 5 per group) is shown. C, D Tumors from mice treated in (A) and (B) were processed to measure miR‐182‐3p expression by TaqMan qPCR. E Representative images of IHC analysis of the indicated markers on tumor samples from mice bearing MDA‐MB‐231 human breast cancer xenografts. Scale bar: 50 μm. F The histograms show the expression of TRF2, calculated as immunoreactivity score (IRS) by IHC, and the count of positive cells to γH2AX, TUNEL or CD31 staining. The analyses were performed on three mice per group, and the points represent the number of field analyzed for each condition. G, H Luminescent MDA‐MB‐436 cells were injected into the brain and monitored by IVIS imaging system. After 1 week from implant, treatment with LNPs‐miR‐Control and LNPs‐miR‐182‐3p was performed as indicated in (A) and (B). Representative images from in vivo (upper panel) or ex‐vivo (bottom panel) brain tumors are shown in (G). Boxplots (H) show the measurement of photons for each brain tumor ( n = 5 per group) acquired at the indicated times. Data information: For (A, B, F), data are shown as mean ± SD. For (C, D, H), the line in the middle of the box plot denotes a median value, the limits of box represent the interquartile range (25 th to 75 th percentiles), while, the whiskers denote the minimum to maximum values. For (A–D) and (H), P values are determined by unpaired two‐tailed t‐ test; for (F), P values are determined by Mann–Whitney t ‐test. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, TUNEL Assay, Staining, Injection, Imaging, In Vivo, Ex Vivo, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: The organs (brain, liver, kidney) taken from mice, previously engrafted with MDA‐MB‐231 cells and treated with LNPs‐empty, LNPs‐miR‐Control or LNPs‐miR‐182‐3p, were assayed for miR‐182‐3p expression by TaqMan qPCR. Representative images show IHC analysis on tumor samples, from mice bearing MDA‐MB‐436 human breast cancer xenografts, with the indicated markers. Scale bar: 50 μm. The histograms show the expression of TRF2 indicated as immunoreactivity score (IRS) and the percentage of positive cells to γH2AX, TIUNEL or CD31 staining in MDA‐MB‐436 xenografts. Three mice per group were analyzed, the points represent the number of field analyzed for each condition. Data information: For (A, C), data are presented as mean values ± SD. Statistical significance using unpaired (A) or Mann–Whitney t‐ test (C) was calculated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B PDTCs #1 and #2 underwent two rounds of transfection with miR‐Control or miR‐182‐3p. Three days after the second transfection, miR‐182‐3p and TRF2 expression were analyzed by TaqMan qPCR and western blotting, respectively. Actin was used as loading control. C, D Left panel, area of each PDTCs was measured by ImageJ. Right panel, representative images are shown. Scale bar: 50 μm. At least 85 3D cells were analyzed for each experimental condition. E NSG mice implanted with breast PDTX (#2) were treated with LNPs‐empty, LNPs‐miR‐Control or LNPs‐miR‐182‐3p as indicated in the scheduling. Caliper measurement of tumors was taken at the indicated days. The mean of tumor volumes ( n = 5 per group) is shown. F miR‐182‐3p expression of tumors from mice treated in (E) was assayed by TaqMan qPCR. G Representative images of IHC analysis of the indicated markers from tumors of the experiment showed in (E). Scale bar: 50 μm. H The histograms show the expression levels of TRF2 measured as immunoreactivity score (IRS), the percentage of positive cells to γH2AX and TUNEL. The analysis was performed on three mice per group, the points represent the number of field analyzed for each condition. Data information: For (A–F) and (H), data are shown as mean ± SD. For (A–F), P values are determined by unpaired two‐tailed t‐ test; for (H), P values are determined by Mann–Whitney t ‐test. For the experiments showed in (A, B) and (C, D) two or three biological replicates were performed, respectively. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Expressing, Western Blot, TUNEL Assay, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Representative images of intestine sections from mice previously treated with LNPs‐Empty or LNPs‐miR‐182‐3p. H&E staining (scale bar: 200 μm) and IHC analysis with TRF2 or γH2AX antibodies are shown (scale bar: 50 μm). B, C Quantification of TRF2 expression as immunoreactivity score (IRS) (B) and of γH2AX‐positive cells (%) (C) on intestine samples. D Representative H&E (scale bar: 200 μm), TRF2 and γH2AX images of skin samples corresponding to LNPs‐Empty or LNPs‐miR‐182‐3p treated animals (scale bar: 50 μm). E, F Quantification of TRF2 expression as immunoreactivity score (IRS) (E) and of γH2AX‐positive cells (%) (F) on skin samples. G Representative H&E (scale bar: 200 μm), TRF2 and γH2AX images of bone marrow samples corresponding to LNPs‐Empty or LNPs‐miR‐182‐3p treated animals (scale bar: 50 μm). H, I Quantification of TRF2 expression as immunoreactivity score (IRS) (H) and of γH2AX‐positive cells (%) (I) on bone marrow samples. Data information: For (B, C, E, F, H, I), data are shown as mean ± SD. A Mann–Whitney test t‐ test was used to calculate statistical significance. Four mice per group were analyzed, the points represent the number of field analyzed for each condition. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Staining, Expressing, MANN-WHITNEY

Journal: Journal of Cell Science

Article Title: Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

doi: 10.1242/jcs.219311

Figure Lengend Snippet: Biphasic TRF2 recruitment to non-telomeric damage sites in nuclei of HeLa cells. (A) PAR stimulation by PARG inhibition (PARGi) promotes GFP–TRF2 accumulation at low input-power damage sites (indicated by arrowheads). Box plot shows quantification of the relative increase of GFP signals at damage sites. (B) Time-course analysis of GFP–TRF2 recruitment to laser-induced DNA damage sites (between arrowheads). (C) Quantification of GFP signals at damage sites in B. N=16. (D) Detection of endogenous TRF2 at damage sites. PARP inhibition (PARPi) suppresses phase I, but has no effect on phase II, TRF2 recruitment. (E) Quantification of the effects of PARP inhibitors (NU1025 and olaparib) on immediate (1 min, phase I) and late (30 min, phase II) GFP–TRF2 recruitment. (F) Time course analysis of the effect of IDP depletion on dispersion of TRF2 at damage sites in HeLa cells transfected with control siRNA (siControl) or FET siRNAs (siFET). Left: quantification of signal intensity changes of GFP–TRF2 (blue) and dark line (red). In box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers represent the lowest datum still within 1.5× IQR (inter-quartile range) of the lower quartile, and the highest datum still within 1.5× IQR of the upper quartile. Scale bar: 10 μm.

Article Snippet: Mouse monoclonal antibodies specific for PAR polymers [BML-SA216–0100, Enzo Life Sciences; 1:500 dilution for immunofluorescence staining (IF)], TRF2 (NB100–56506, Novus Biologicals; 1:500 for IF), MRE11 [GTX70212, GeneTex; 1:500 for IF, 1:1000 for western blotting (WB)], GFP (632592, Takara Bio; 1:500 for ChIP), Actin (A4700, Sigma; 1:1000 for WB), FLAG (F3165, Sigma; 1:1000 for WB), and BRCA1 (GTX70111, GeneTex; 1:1000 for WB), as well as rabbit polyclonal antibodies specific for γH2AX (GTX628789, GeneTex; 1:500 for IF), EWSR1 (GTX114069, GeneTex; 1:1000 for WB), TAF15 (GTX103116, GeneTex; 1:1000 for WB), H3 (14-411, Upstate Bio; 1:1000 for WB) and Rad21 ( Kong et al., 2014 ; 1:2000 for WB) were used.

Techniques: Inhibition, Dispersion, Transfection, Control

Journal: Journal of Cell Science

Article Title: Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

doi: 10.1242/jcs.219311

Figure Lengend Snippet: Distinct TRF2 domain requirement for phase I and II recruitment. (A) Schematic diagrams of TRF2 deletion mutants. (B) Time course analysis of damage site localization (between arrowheads) of wild-type and TRF2 deletion mutants in the nuclei of HeLa cells. (C) Box plot shows quantification of TRF2 mutant GFP signals at damage sites at 1 min (phase I) and 30 min (phase II) post-damage induction. (D) Box plot shows quantification on the effects of the N-terminal amino acid substitutions on phase I recruitment of TRF2. Arginine-to-alanine mutations (RA), arginine-to-lysine substitution (RK) and the HJ binding mutation (H31A) were tested. WT, wild type. Amino acid sequences of N-terminal domain mutations are shown on the right. In box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers represent the lowest datum still within 1.5× IQR (inter-quartile range) of the lower quartile, and the highest datum still within 1.5× IQR of the upper quartile. Scale bar: 10 μm.

Article Snippet: Mouse monoclonal antibodies specific for PAR polymers [BML-SA216–0100, Enzo Life Sciences; 1:500 dilution for immunofluorescence staining (IF)], TRF2 (NB100–56506, Novus Biologicals; 1:500 for IF), MRE11 [GTX70212, GeneTex; 1:500 for IF, 1:1000 for western blotting (WB)], GFP (632592, Takara Bio; 1:500 for ChIP), Actin (A4700, Sigma; 1:1000 for WB), FLAG (F3165, Sigma; 1:1000 for WB), and BRCA1 (GTX70111, GeneTex; 1:1000 for WB), as well as rabbit polyclonal antibodies specific for γH2AX (GTX628789, GeneTex; 1:500 for IF), EWSR1 (GTX114069, GeneTex; 1:1000 for WB), TAF15 (GTX103116, GeneTex; 1:1000 for WB), H3 (14-411, Upstate Bio; 1:1000 for WB) and Rad21 ( Kong et al., 2014 ; 1:2000 for WB) were used.

Techniques: Mutagenesis, Binding Assay

Journal: Journal of Cell Science

Article Title: Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

doi: 10.1242/jcs.219311

Figure Lengend Snippet: Phase II recruitment is affected by TERT and is dependent on the iDDR region in the hinge domain of TRF2. (A) Box plot shows that TERT depletion using siRNA inhibits phase II recruitment of TRF2 to DNA damage sites in nuclei of HeLa cells. (B) Schematic diagrams of chimeric TRF1/2 mutants (as previously described in Okamoto et al., 2013) used in the experiments represented in panels C–F. (C) Left: representative cell images of the recruitment of chimeric mutants to damage sites (between arrowheads) at ∼1 min (phase I) and ∼30 min (phase II) after damage induction. Right: box plots show quantification of the GFP–TRF2 signal increase at phase I and phase II at damage sites. (D) Comparison of the GFP signal at damage sites in HeLa cells expressing iDDR and TIN2 deletion mutants at 30 min after damage induction. (E) The effect of MRE11 and NBS1 siRNA (siMRE11 and siNBS1) depletion on phase I and II recruitment of GFP–TRF2 was examined comparing to control siRNA (siControl). HeLa cells were fixed and stained with anti-MRE11 antibody (red) to confirm the depletion. Box plot shows quantification of the GFP–TRF2 signal increase at damage sites in control or MRE11 and NBS1 siRNA-treated cells. (F) ChIP-qPCR analysis of GFP–TRF2 binding at I-PpoI cut sites. TRF2 binding was examined in the absence or presence of I-PpoI, and with and without MMS as indicated. Cells were further treated with DMSO or PARP inhibitor (PARPi) (left panel). Cells expressing GFP only were used as a negative control. Alternatively, cells were transfected with control (siCon) or MRE11 and NBS1 siRNA in the presence of I-PpoI with or without MMS (right panel). In box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers represent the lowest datum still within 1.5× IQR (inter-quartile range) of the lower quartile, and the highest datum still within 1.5× IQR of the upper quartile. Bar graphs show mean±s.d., *P<0.01, **P<0.001, ***P<0.0001. Scale bars: 10 μm.

Article Snippet: Mouse monoclonal antibodies specific for PAR polymers [BML-SA216–0100, Enzo Life Sciences; 1:500 dilution for immunofluorescence staining (IF)], TRF2 (NB100–56506, Novus Biologicals; 1:500 for IF), MRE11 [GTX70212, GeneTex; 1:500 for IF, 1:1000 for western blotting (WB)], GFP (632592, Takara Bio; 1:500 for ChIP), Actin (A4700, Sigma; 1:1000 for WB), FLAG (F3165, Sigma; 1:1000 for WB), and BRCA1 (GTX70111, GeneTex; 1:1000 for WB), as well as rabbit polyclonal antibodies specific for γH2AX (GTX628789, GeneTex; 1:500 for IF), EWSR1 (GTX114069, GeneTex; 1:1000 for WB), TAF15 (GTX103116, GeneTex; 1:1000 for WB), H3 (14-411, Upstate Bio; 1:1000 for WB) and Rad21 ( Kong et al., 2014 ; 1:2000 for WB) were used.

Techniques: Comparison, Expressing, Control, Staining, ChIP-qPCR, Binding Assay, Negative Control, Transfection

Journal: Journal of Cell Science

Article Title: Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

doi: 10.1242/jcs.219311

Figure Lengend Snippet: TRF2 specifically promotes non-sister chromatid HR repair. (A) The effect of TRF2 depletion on DSB repair using the I-SceI HR system. Complementation analysis of TRF2-depleted cells was performed using the wild type and chimeric TRF1/2 mutants. BRCA1 depletion was used as a positive control. Comparable expression levels of the recombinant TRF2 proteins were confirmed using western blot analysis (right). Histone H3 serves as a loading control. (B) The effect of TRF2 depletion on different DSB repair pathways was examined using SCE, classic NHEJ (C-NHEJ) and alternative NHEJ (Alt-NHEJ) assays. (C) Schematic showing similarity between strand invasion in D-loop formation at telomeres and at DSB sites by TRF2. (D) Schematic showing biphasic mechanism of TRF2 recruitment to damage sites. Phase I involves PARP-dependent recruitment through the basic domain. Phase II is mediated by the MYB/SANT domain, which is also dependent on the iDDR region and the Mre11 complex. Bar graphs show mean±s.d.

Article Snippet: Mouse monoclonal antibodies specific for PAR polymers [BML-SA216–0100, Enzo Life Sciences; 1:500 dilution for immunofluorescence staining (IF)], TRF2 (NB100–56506, Novus Biologicals; 1:500 for IF), MRE11 [GTX70212, GeneTex; 1:500 for IF, 1:1000 for western blotting (WB)], GFP (632592, Takara Bio; 1:500 for ChIP), Actin (A4700, Sigma; 1:1000 for WB), FLAG (F3165, Sigma; 1:1000 for WB), and BRCA1 (GTX70111, GeneTex; 1:1000 for WB), as well as rabbit polyclonal antibodies specific for γH2AX (GTX628789, GeneTex; 1:500 for IF), EWSR1 (GTX114069, GeneTex; 1:1000 for WB), TAF15 (GTX103116, GeneTex; 1:1000 for WB), H3 (14-411, Upstate Bio; 1:1000 for WB) and Rad21 ( Kong et al., 2014 ; 1:2000 for WB) were used.

Techniques: Positive Control, Expressing, Recombinant, Western Blot, Control

Journal: Journal of Nucleic Acids

Article Title: A ssDNA Aptamer That Blocks the Function of the Anti-FLAG M2 Antibody

doi: 10.4061/2011/720798

Figure Lengend Snippet: 3XABA oligonucleotide blocks the interaction of Flag-tagged proteins with the M2 antibody. (a)-(b) The 3XABA oligo blocks the binding of Flag-TRF2 ΔB to M2-coated beads. Magnetic beads coated with the M2 antibody were incubated in the absence (No Comp) or presence of the indicated competitor (3XABA, 3XCTR, 3XFLAG). In vitro translated [ 35 S]-labeled Flag-TRF2 ΔB was then added and the amount captured by the beads was determined by SDS-PAGE electrophoresis and exposure to a PhophorImager cassette (a). In a second experiment done in triplicate, the amount of [ 35 S]-labeled protein captured was counted by scintillation (b). The amount of [ 35 S]-labeled protein captured in the absence of competitor (No Comp) was arbitrarily set to 100%. In both experiments, beads coated with normal mouse IgG were included as negative control for the capture (IgG). Data represent the mean ± S.D. ( n = 3). (c)-(d) The 3XABA oligo elutes the Flag-TRF2 ΔB proteins already bound to M2-coated beads. The [ 35 S]-Flag-TRF2 ΔB protein was first captured by magnetic beads coated with the M2 antibody. The beads were then incubated in the absence (No comp) or presence of the indicated competitor (3XABA, 3XCTR, 3XFLAG). The amount of [ 35 S]-Flag-TRF2 ΔB released was determined by SDS-PAGE electrophoresis and exposure to a PhophorImager cassette (c). In a second experiment done in triplicate, the amount of [ 35 S]-labeled protein released was counted by scintillation (d). The amount of [ 35 S]-labeled protein released by the boiling (total) was arbitrarily set to 100%. In both experiments, beads boiled to release to all of the captured [ 35 S]-labeled protein were included as positive control for the elution (Total). Data represent the mean ± S.D. ( n = 3).

Article Snippet: Normal mouse IgG (cat. # sc-2025) and anti-vimentin mouse monoclonal antibody (IgG 1 clone sc-6260) were obtained from Santa Cruz (Santa Cruz, CA), as was the rabbit polyclonal antibody against TRF2 (cat. # H-300).

Techniques: Binding Assay, Magnetic Beads, Incubation, In Vitro, Labeling, SDS Page, Electrophoresis, Negative Control, Positive Control