Journal: Nature structural & molecular biology

Article Title: The macroH2A1.2 histone variant links ATRX loss to alternative telomere lengthening.

doi: 10.1038/s41594-019-0192-3

Figure Lengend Snippet: (A) ChIP for γ-H2AX at the indicated loci in K562 cells in the presence (WT) or absence of macroH2A1.2 (1.2 CRISPR-KO). Samples were normalized to untreated WT samples, values represent mean and s.d. from 3 independent experiments. See for macroH2A1.2 ChIP. (B) γ-H2AX ChIP at the indicated loci in U2OS cells treated with vehicle (DMSO) or HU in the presence or absence of ATRX re-expression. Values represent mean and s.d. from 3 independent experiments. (C) Frequency of TIFs in U2OS cells with ATRX induction in the presence or absence of Aph, TIFs were defined based on co-localization of γ-H2AX (red) and TRF2 (green), a representative image from Aph-treated, sh-RFP expressing U2OS cells is shown; scale bar = 5 μm. Box plots depict the number of TIFs per cell. N: number of cells. *** p < 10 −8 by Mann-Whitney U test, one of two independent experiments is shown. (D) Model linking ATRX and macroH2A1.2 to ALT telomere maintenance. ATRX-dependent macroH2A1.2 retention at stalled replication forks protects from excessive DNA damage in ALT-negative cells. In ALT-positive cells, ATRX deficiency leads to macroH2A1.2 loss and DSBs in response to RS, which triggers DDR-dependent macroH2A1.2 re-deposition to facilitate HR. In the absence of RS, lack of ATRX has little effect on telomeric macroH2A1.2 levels, pointing to DNA damage-induced modulation of ATRX function, which may involve ATRX phosphorylation in S phase (see ) .

Article Snippet: The following antibodies were used for ChIP: α-macroH2A1.2 (Millipore MABE61), α-phospho-S139-H2AX (Millipore 05–636), α-H2B (Abcam ab52484), α-H2A (Abcam ab18255), TRF2 (Novus Biologicals IMG-124A) and normal mouse IgG (Millipore 12–371).

Techniques: CRISPR, Expressing, MANN-WHITNEY

Journal: Oncotarget

Article Title: ERK1/2/MAPK pathway-dependent regulation of the telomeric factor TRF2

doi: 10.18632/oncotarget.10316

Figure Lengend Snippet: A. Equimolar amounts of GST, GST-ELK and His-TRF2 were incubated in the absence (−) or presence (+) of recombinant active ERK2. The phosphorylated proteins were detected after SDS PAGE using a specific anti-PX[phospho]SP antibody (pPXSP). Coomassie blue staining of the membrane is shown as a loading control. B. Alignment of TRF2 sequences from different mammalian species show the conservation of a MAPK phosphorylation consensus PXSP target site. The species and respective Genbank reference numbers corresponding to the sequences are reported. The conserved PXSP site is shown in bold and the conserved S residue underlined. C. Specificity of the immune serum using peptides containing phospho-S323. His-TRF2 was phosphorylated or not by recombinant active ERK2. The same amounts of proteins were submitted to immunoblotting analysis with the anti-pS323 antibody (pTRF2). Coomassie blue staining is shown as a loading control. D. A375 cells were stably transfected with WT-TRF2 or TRF2 S323A . Cells were treated (+) or not (−) with PD184352 (PD). Phosphorylated TRF2 was immunoprecipitated with the specific anti-pTRF2 antibody and detected by immunoblotting with an anti-TRF2 antibody (IP). Total TRF2 is shown as a loading control (input) and pERK1/2 as a control of PD184352 activity.

Article Snippet: Immuno-precipitation assays were performed on protein G sepharose beads with either 3 μg of the polyclonal phospho-TRF2 antibody or 2 μg of a TRF2 antibody (Imgenex IMG-124A) on 200 μg to 1 mg proteins, incubated for 16 hours at 4°C.

Techniques: Incubation, Recombinant, SDS Page, Staining, Membrane, Control, Phospho-proteomics, Residue, Western Blot, Stable Transfection, Transfection, Immunoprecipitation, Activity Assay

Journal: Oncotarget

Article Title: ERK1/2/MAPK pathway-dependent regulation of the telomeric factor TRF2

doi: 10.18632/oncotarget.10316

Figure Lengend Snippet: A. Immortalized BJ fibroblasts were serum deprived for 24 hours. BJ cells were then stimulated with 10% FCS for the indicated times. Phosphorylated forms of TRF2 were immunoprecipitated with the specific anti-pTRF2 antibodies and detected by immunoblotting with an anti-TRF2 antibody (IP p-TRF2). Total TRF2 and Hsp-90 are shown as loading controls and the phosphorylated forms of ERK1/2 as a control of serum-dependent activation of ERK1/2 (Input). B. Different tumor cells were tested for the presence of pTRF2 by immunoprecipitation in the presence (+) or absence (−) of PD184352 (PD) (Cal33, BJ-Ras (BJ-R), A375, U2OS). A short (Short exp.) and long (Long exp.) exposure of the blots are shown (IP p-TRF2). Total TRF2, Hsp90 and tubulin are shown as loading controls and the phosphorylated forms of ERK1/2 as a control of PD184352 activity (Input).

Article Snippet: Immuno-precipitation assays were performed on protein G sepharose beads with either 3 μg of the polyclonal phospho-TRF2 antibody or 2 μg of a TRF2 antibody (Imgenex IMG-124A) on 200 μg to 1 mg proteins, incubated for 16 hours at 4°C.

Techniques: Immunoprecipitation, Western Blot, Control, Activation Assay, Activity Assay

Journal: Oncotarget

Article Title: ERK1/2/MAPK pathway-dependent regulation of the telomeric factor TRF2

doi: 10.18632/oncotarget.10316

Figure Lengend Snippet: A. In situ proximity ligation assay (PLA) with anti-TRF2 and anti-pERK1/2 antibodies alone (negative controls, but in the presence of the two secondary antibodies) or in combination (TRF2 + pERK1/2) in A375 cells in the presence of DMSO or PD184352 (scale bars represent 10μm). A 3D image reconstruction after confocal microscopy imaging of PLA with a combination of anti-TRF2 and anti-pERK1/2 antibodies is also shown (scale bar represents 1μm). B. PLA with combined anti-TRF2 and anti-pERK antibodies in normal human skin tissue or cutaneous squamous cell carcinoma, normal lung or lung squamous cell carcinoma and normal cervix or cervical squamous cell carcinoma (scale bars represent 35μm). A 3D image reconstruction after confocal microscopy imaging of PLA with a combination of anti-TRF2 and anti-pERK1/2 antibodies in cutaneous squamous cell carcinoma is also shown (scale bar represents 1μm).

Article Snippet: Immuno-precipitation assays were performed on protein G sepharose beads with either 3 μg of the polyclonal phospho-TRF2 antibody or 2 μg of a TRF2 antibody (Imgenex IMG-124A) on 200 μg to 1 mg proteins, incubated for 16 hours at 4°C.

Techniques: In Situ, Proximity Ligation Assay, Confocal Microscopy, Imaging

Journal: Oncotarget

Article Title: ERK1/2/MAPK pathway-dependent regulation of the telomeric factor TRF2

doi: 10.18632/oncotarget.10316

Figure Lengend Snippet: A. A375, SKMel-51 and BJ-RAS cells were incubated in the presence of 50 μg/ml cycloheximide (CHX) for 16 hours in the presence or absence of PD184352 (PD). Total TRF2 and tubulin amounts were evaluated by immune-blotting. Tubulin and coomassie blue staining of the membrane (B) are shown as loading controls. B. Densitometric quantifications of the blots shown in A. The TRF2 expression level was normalized with three to four different loading controls and expressed relative to the control conditions (results are expressed as mean ± SD). One way ANOVA statistical analysis is included: * p<0.05; ** p<0.01; *** p<0.001). C. A375 cells over-expressing either TRF2-WT or TRF2-S323A were incubated in the presence of 50 μg/ml cycloheximide for 16 hours. Tubulin is shown as loading control. Densitometric quantification of the blot is shown (results are expressed as mean ± SD. One way ANOVA statistical analysis is included: * p<0.05; ** p<0.01; *** p<0.001).

Article Snippet: Immuno-precipitation assays were performed on protein G sepharose beads with either 3 μg of the polyclonal phospho-TRF2 antibody or 2 μg of a TRF2 antibody (Imgenex IMG-124A) on 200 μg to 1 mg proteins, incubated for 16 hours at 4°C.

Techniques: Incubation, Staining, Membrane, Expressing, Control

Journal: Oncotarget

Article Title: ERK1/2/MAPK pathway-dependent regulation of the telomeric factor TRF2

doi: 10.18632/oncotarget.10316

Figure Lengend Snippet: A. Confocal imaging of the co-staining of 53BP1 by immunofluorescence (red) and telomeres by fluorescent in situ hybridization (TelC-FITC, green) in A375 cells were the expression of WT-TRF2, TRF2 S323A or TRF2-ΔBΔM was induced by tetracycline (Tet) treatment. Colocalisation events were counted as Telomere dysfunction-Induced Foci (TIF, indicated with white arrows, scale bars represent 5μM) and the proportion of nuclei showing more than 3 TIF is indicated (right panel, results are expressed as mean ± SD, t-test statistical analysis is included: * p<0.05; ** p<0.01; *** p<0.001). B. The proportion of cells in each phase of the cell cycle was determined by DNA labeling with propidium iodide and FACS analysis. Sub G1 stands for cells with fragmented DNA, a hallmark of apoptosis. C. Conditional overexpression of different forms of TRF2 (WT, TRF2 S323A and TRF2 ΔBΔM ) was induced by tetracycline (Tet) in A375 cells. Seven days after tetracycline stimulation, cells were colored with giemsa blue. A close-up in TRF2 S323A overexpression well shows the few remaining cells at the end of the experiment. D. The cells were tested for b-galactosidase activity after seven days of tetracyclin induction (lower pictures). The percentage of β-galactosidase positive cells under tetracycline-induced conditions is specified below the images. E. Seven days after induction of the different forms of TRF2 by tetracycline (+), cells were tested for the presence of phosphorylated forms of p53. Actin and p53 are shown as loading controls.

Article Snippet: Immuno-precipitation assays were performed on protein G sepharose beads with either 3 μg of the polyclonal phospho-TRF2 antibody or 2 μg of a TRF2 antibody (Imgenex IMG-124A) on 200 μg to 1 mg proteins, incubated for 16 hours at 4°C.

Techniques: Imaging, Staining, Immunofluorescence, In Situ Hybridization, Expressing, DNA Labeling, Over Expression, Activity Assay

Journal: Oncotarget

Article Title: ERK1/2/MAPK pathway-dependent regulation of the telomeric factor TRF2

doi: 10.18632/oncotarget.10316

Figure Lengend Snippet: A. Control A375 cells or A375 cells conditionally expressing WT-TRF2 or TRF2 S323A were subcutaneously injected into nude mice. Doxycycline was added to the drinking water ten days after injection to induce the transgenes expression. The tumor volume is shown and results are expressed as mean ± SD (t-test statistical analysis is included: * p<0.05; ** p<0.01; *** p<0.001). B. Immunoblots showing the expression of TRF2 and actin (loading control) in tumor extracts prepared at the end of the tumor xenograft experiment.

Article Snippet: Immuno-precipitation assays were performed on protein G sepharose beads with either 3 μg of the polyclonal phospho-TRF2 antibody or 2 μg of a TRF2 antibody (Imgenex IMG-124A) on 200 μg to 1 mg proteins, incubated for 16 hours at 4°C.

Techniques: Control, Expressing, Injection, Western Blot