Journal: Genome Biology

Article Title: A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression

doi: 10.1186/s13059-015-0791-1

Figure Lengend Snippet: TERT localizes to telomeres in S but not G1 phase of the cell cycle. a IF analysis of fixed HeLa cells expressing FLAG-SNAP-TERT, synchronized in G1 and S phase of the cell cycle (scale bar = 5 μm). Cells expressing FLAG-SNAP-TERT displayed telomere-localized TERT foci in S but not G1 phase of the cell cycle, while parental cells never showed telomere-localized TERT foci. b FACS analysis of the DNA content of cells synchronized in S phase showed a peak between the 2 N and 4 N peaks of asynchronous cells, confirming that they were in S phase. The G1 cell population contained 2 N and 4 N peaks but was depleted for cells with intermediate DNA content. 4 N cells, which failed to release from their mitotic arrest, were easily distinguished from G1 cells by their morphology. c Quantification of the number of TERT foci which co-localized with TRF2 signals in edited HeLa cells synchronized at different stages of the cell cycle. Data were generated from two independent experiments, each analyzing 50 cells per condition (mean ± standard deviation). d FACS analysis of the DNA content of edited HeLa cells released from a double thymidine block as they transition through S phase. Prior to release, the cell population contained mostly cells with 2 N DNA content, which progressively increased as the cells underwent DNA replication. Nine to ten hours after release, DNA replication was complete, as indicated by the majority of cells having 4 N DNA content. e Quantification of the number of TERT foci co-localized with TRF2 signals at different time points during S phase (50 cells per time point, mean ± standard error of the mean; for corresponding images see Fig. S4 in Additional file ). A.U. arbitrary units, Propidium Iodide (PI)

Article Snippet: Cells were then re-permeabilized using Triton X Buffer for 10 min at room temperature, and incubated in blocking buffer (3 % BSA in PBS) for 30 min. After blocking, cells were incubated with primary antibodies for TRF2 (Imgenex, IMG-124A, 1:500) and coilin (Santa Cruz, sc-32860, 1:100) in blocking buffer for 1 h. Next, cells were washed with PBS and incubated with secondary antibodies (Life Technologies, A-31556, and Abcam, ab150117, 1:500) in blocking buffer for 1 h. After a final wash, cells were mounted using ProLong® Diamond Antifade Mountant (Life Technologies, P36970).

Techniques: Expressing, Generated, Standard Deviation, Blocking Assay

Journal: Journal of Biological Chemistry

Article Title: Androgen Receptor Interacts with Telomeric Proteins in Prostate Cancer Cells

doi: 10.1074/jbc.m109.098798

Figure Lengend Snippet: FIGURE 1. Casodex disrupts telomeric complexes in AR-positive prostate cancer cells. A and B, LNCaP cells treated with 100 M Casodex for 48 h (A and B) or with 20 g/ml etoposide for 1 h (B) were co-immunostained with antibodies against 53BP1 and TRF2. LNCaP (C) or PC-3 (D) cells were treated with or without 100 M Casodex for 48 h and then immunostained with 53BP1 antibody. 53BP1 foci were counted, and data are presented as the percentage of cells with 0–5, 6–10, 11–20, or 20 foci/cell. Immunostaining and confocal microscopy were performed as described under “Experimental Procedures.” 80 cells in each treatment group were scored in three separate experiments.

Article Snippet: For Western blot analysis, membraneswere probedwith antibodies against AR (AR-N20, Santa Cruz Biotechnology), TRF2 (IMG-124A), TRF1 (H-242, Santa Cruz Biotechnology), HA (sc-805, Santa Cruz Biotechnology), TIN2 (15), TPP1 (Abcam), or glyceraldehyde-3-phosphate dehydrogenase (Chemicon).

Techniques: Immunostaining, Confocal Microscopy

Journal: Journal of Biological Chemistry

Article Title: Androgen Receptor Interacts with Telomeric Proteins in Prostate Cancer Cells

doi: 10.1074/jbc.m109.098798

Figure Lengend Snippet: FIGURE 2. Casodex disrupts telomeric complexes without affecting the expression of telomeric proteins. A, LNCaP cells treated with 100 M Casodex for 0, 3, 10, 24, or 48 h were immunostained for 53BP1. 53BP1 foci were counted, and cells were categorized as having 5 or 5 foci/cell. 200 cells in each treatment group were scored in three independent experiments. B, total RNA from LNCaP cells treated with Casodex was extracted using TRIzol (Invitrogen), and RT-PCR was performed as described previously (32) to measure mRNA levels. Sequence-specific primers for TIN2, TRF1, TRF2, TPP1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously (11), and for AR, 5-tcagttcacttttgacctgctaa-3 (forward) and 5-gtggaaatagat- gggcttga-3 (reverse) primers were used. C, whole cell lysates of LNCaP cells treated with Casodex were sub- jected to Western blot analysis to determine TIN2, TRF1, TRF2, AR, and glyceraldehyde-3-phosphate dehydro- genase (loading control) protein levels.

Article Snippet: For Western blot analysis, membraneswere probedwith antibodies against AR (AR-N20, Santa Cruz Biotechnology), TRF2 (IMG-124A), TRF1 (H-242, Santa Cruz Biotechnology), HA (sc-805, Santa Cruz Biotechnology), TIN2 (15), TPP1 (Abcam), or glyceraldehyde-3-phosphate dehydrogenase (Chemicon).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: Androgen Receptor Interacts with Telomeric Proteins in Prostate Cancer Cells

doi: 10.1074/jbc.m109.098798

Figure Lengend Snippet: FIGURE 4. AR is associated with telomeric proteins in LNCaP cells. A, telomeric proteins are overexpressed in prostate cancer cells. Whole cell lysates prepared from prostate cancer cells (LNCaP, PPC-1, and PC-3) and normal prostate epithelial cells (PrEC) were subjected to Western blot analysis to determine TRF1, TRF2, TIN2, AR, and -actin (loading control) protein levels. B, AR and TRF2 in LNCaP cell lysates were individually immunoprecipitated (IP) using monoclonal antibodies, and immunopre- cipitates and starting lysate (2% of input) were subjected to Western blot (WB) analysis of AR, TRF1, and TRF2. C, cell lysates were prepared from LNCaP cells transfected with HA-tagged TRF1 as described previously (13), and HA-TRF1 in cell lysates was immunoprecipitated using antibodies against the HA epitope. Immunoprecipitates and unprecipitated lysate (10% of input) were subjected to Western blot analysis of AR and TIN2. D, sucrose density gradient analysis of telomeric proteins and AR in LNCaP cells. Nuclear extract (NE) prepared from exponentially growing LNCaP cells was subjected to sucrose density gradient centrifugation, and the gradient was resolved into 12 fractions, which were subjected to Western blot analysis to identify the distribution of TRF1, TRF2, TIN2, TPP1, and AR in the gradient. Lamin B was used as a control. Top, top of the gradient; Bottom, bottom of the gradient; NE, Nuclear extract loaded onto the gradient.

Article Snippet: For Western blot analysis, membraneswere probedwith antibodies against AR (AR-N20, Santa Cruz Biotechnology), TRF2 (IMG-124A), TRF1 (H-242, Santa Cruz Biotechnology), HA (sc-805, Santa Cruz Biotechnology), TIN2 (15), TPP1 (Abcam), or glyceraldehyde-3-phosphate dehydrogenase (Chemicon).

Techniques: Western Blot, Control, Immunoprecipitation, Bioprocessing, Transfection, Gradient Centrifugation

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