Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: Data collection and refinement statistics.

Article Snippet: The equilibrium dissociation constants of the WT and mutant TRF2 TRFH –MCPH1 TBM interactions were determined using a MicroCal iTC 200 calorimeter (Malvern Panalytical).

Techniques: Solvent

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: a Schematic representation of human TRF2 and MCPH1 domains, showing the interaction domains. b Comparison of MCPH1 TBM sequence with those of known TRF2-interacting protein. The conserved amino acid Y/H-X-L-X-P consensus sequence is highlighted. c Dimeric TRF2–MCPH1 structure is shown in a ribbon representation (TRF2, green/cyan; MCPH1, magenta/yellow). d TRF2 and MCPH1 are depicted in green and yellow, respectively, and the residues involved in their interaction are shown. Hydrogen bonding: magenta dashed lines. e MCPH1 TBM (in yellow) is buried inside a hydrophobic pocket formed by TRF2 helices α2 and α3 (in green). f ITC measurement of the interactions between TRF2 TRFH and different MCPH1 TBM mutant peptides. S333phos is a phosphorylated S333 peptide synthesized using a phosphorylated serine as starting material. Equilibrium dissociation constant ( K D ) values derived from ITC data are shown in Table .

Article Snippet: The equilibrium dissociation constants of the WT and mutant TRF2 TRFH –MCPH1 TBM interactions were determined using a MicroCal iTC 200 calorimeter (Malvern Panalytical).

Techniques: Comparison, Sequencing, Mutagenesis, Synthesized, Derivative Assay

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: Equilibrium dissociation constant ( K D ) values.

Article Snippet: The equilibrium dissociation constants of the WT and mutant TRF2 TRFH –MCPH1 TBM interactions were determined using a MicroCal iTC 200 calorimeter (Malvern Panalytical).

Techniques:

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: a Schematic representation of the human MCPH1 constructs generated. The TBM sequence for each construct is shown and amino acid substitutions are depicted in red. b Co-IP with anti-Myc antibody-conjugated agarose beads from lysates of 293T cells expressing Myc-tagged TRF2 and either FLAG-tagged WT MCPH1, FLAG-MCPH1 TBM mutants or FLAG-MCPH1 ΔBRCT . γ-tubulin was used as a loading control. The blot shown is representative of four independent experiments. c Immunostaining-PNA FISH in HeLa cells overexpressing either empty vector or one of the FLAG-tagged WT MCPH1, MCPH1 AA , MCPH1 S333A , MCPH1 S333D , MCPH1 ΔBRCT constructs and either empty vector or HA-TPP1 ΔRD . MCPH1 proteins were detected using an anti-FLAG antibody (green) while telomeres were detected with a Cy3-OO-(CCCTAA) 4 PNA probe (red). 4,6-diamidino-2-phenylindole (DAPI) was used to stain nuclei (blue). Representative images from three independent experiments are shown. White arrowheads indicate MCPH1 foci co-localizing with the telomere signals. Scale bar: 5 μm. d Quantification of the percentage of cells with >5 MCPH1-positive foci at telomeres from c . Data represent the mean ± standard deviation (SD) from three independent experiments. At least 200 cells were scored for each sample. Significance was determined with one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. P values are shown. e Comparison of MCPH1 TBM amino acidic sequence across several mammalian species. The conserved residues are highlighted in yellow, while the residues in red differ from the canonical Y/H-X-L-X-P amino acid sequence. f Immunostaining-PNA FISH in MEFs overexpressing either Myc-WT MCPH1 or Myc-MCPH1 ΔBRCT together with either empty vector or FLAG-TIN2 A110R . Myc-MCPH1 proteins were detected with a Myc antibody (green), while telomeres were detected with either a telomeric PNA probe or a FLAG antibody that recognizes FLAG-TIN2 A110R (in red). Nuclei were stained with DAPI (blue). Representative images from three independent experiments. Scale bar: 5 μm. g Quantification of the percentage of cells with >5 MCPH1-positive foci at telomeres from f . Data are representative of the mean of three independent experiments ± SD. A minimum of 200 cells for each sample were scored. Statistical analysis: one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: The equilibrium dissociation constants of the WT and mutant TRF2 TRFH –MCPH1 TBM interactions were determined using a MicroCal iTC 200 calorimeter (Malvern Panalytical).

Techniques: Construct, Generated, Sequencing, Co-Immunoprecipitation Assay, Expressing, Control, Immunostaining, Plasmid Preparation, Staining, Standard Deviation, Comparison

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: a Immunostaining for MCPH1 telomeric localization in MCPH1 +/+ HCT116 and two CRISPR/Cas9 MCPH1 Δ/Δ HCT116 clones (B2 and A5) overexpressing the indicated constructs. MCPH1 localization at telomeres was assessed using an anti-MCPH1 antibody (green) and telomere were detected through PNA-FISH (red). Representative images from either three (MCPH1 +/+ and MCPH1 Δ/Δ B2 + vector, TRF2 ΔBΔM or TPP1 ΔRD ) or two (MCPH1 Δ/Δ A5 and samples with TRF2 ΔBΔM + TPP1 ΔRD ) independent experiments. b Percentage of cells with >5 MCPH1-positive foci at telomeres from ( a ). Data represent mean values ± SD. n = 3 for MCPH1 +/+ and MCPH1 Δ/Δ B2 + vector, TRF2 ΔBΔM and TPP1 ΔRD ; n = 2 for MCPH1 Δ/Δ A5 and samples with TRF2 ΔBΔM + TPP1 ΔRD . A minimum of 200 cells were scored for each sample. c BARD1 TIF analysis in WT and MCPH1 Δ/Δ cells reconstituted with either empty vector, WT MCPH1, MCPH1 S333A or MCPH1 S333D and overexpressing either empty vector or FLAG-TPP1 ΔRD . Representative images from two independent experiments. d Percentage of cells with >5 BARD1-positive TIFs from ( c ). The means from two independent experiments ± SD are shown. At least 200 cells were scored for each sample. e – g Quantification of the percentage of cells with >5 p-RPA32 (S33) ( e ) and CTIP ( f ) TIFs and with >3 EXOI ( g ) TIFs in WT and MCPH1 Δ/Δ cells reconstituted with the indicated constructs and overexpressing either empty vector or FLAG-TPP1 ΔRD (see also Supplementary Fig. ). Data represent the mean ± SD from two independent experiments. At least 200 cells were scored for each sample. h RAD51 TIF analysis in TPP1 ΔRD -expressing U2OS cells treated with either scrambled or MCPH1 shRNA and reconstituted with either empty vector, WT MCPH1, MCPH1 S333A or MCPH1 S333D . Representative images from three independent experiments. i Quantification of the percentage of cells with >5 RAD51-positive TIFs shown in ( h ). Data represent the mean values ± SD, n = 3. At least 200 cells were scored for each sample. The statistical analysis for b , d – g and i was performed using one-way ANOVA followed by Tukey’s multiple comparison test. Scale bars for a , c , h : 5 μm.

Article Snippet: The equilibrium dissociation constants of the WT and mutant TRF2 TRFH –MCPH1 TBM interactions were determined using a MicroCal iTC 200 calorimeter (Malvern Panalytical).

Techniques: Immunostaining, CRISPR, Clone Assay, Construct, Plasmid Preparation, Expressing, shRNA, Comparison

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: a Telomeric PNA-FISH staining of metaphase spreads from MCPH1 +/+ , MCPH1 Δ/Δ B2, and MCPH1 Δ/Δ A5 HCT116 cells overexpressing either empty vector, TRF2 ΔBΔM , TPP1 ΔRD , or both TRF2 ΔBΔM and TPP1 ΔRD . Telomeres were detected using a PNA probe (red) and DAPI was used to stain the chromosomes (blue). Representative images from either three (MCPH1 +/+ and MCPH1 Δ/Δ B2 + vector, TRF2 ΔBΔM , or TPP1 ΔRD ) or two (MCPH1 Δ/Δ A5 and samples with TRF2 ΔBΔM + TPP1 ΔRD ) independent experiments. White and green arrowheads indicate chromosome and chromatid fusions, respectively. Scale bar: 5 μm. b , c Quantification of the percentage of chromosome ( b ) and chromatid ( c ) fusions observed in metaphase spreads shown in ( a ). Data represent the mean values ± SD. n = 3 for MCPH1 +/+ and MCPH1 Δ/Δ B2 + vector, TRF2 ΔBΔM , and TPP1 ΔRD ; n = 2 for MCPH1 Δ/Δ A5 and samples with TRF2 ΔBΔM + TPP1 ΔRD . A minimum of 30 metaphases for each sample were examined per experiment. Significance was determined using one-way ANOVA followed by Tukey’s multiple comparison test. d Representative images of telomere sister chromatid exchanges (T-SCEs) (arrowheads) from three independent CO-FISH experiments on metaphase spreads of U2OS cells expressing TPP1 ΔRD and either scrambled or MCPH1 shRNA. Sister chromatid telomeres were labeled with a FAM-OO-(TTAGGG) 4 PNA probe (green) and with a Cy3-OO-(CCCTAA) 4 PNA probe (red) to detect telomeres generated by leading and lagging strand replication, respectively. Scale bar: 5 μm. e Quantification of the percentage of T-SCEs observed on metaphase spreads of U2OS cells expressing either empty vector or TPP1 ΔRD after MCPH1 depletion and reconstitution with the indicated constructs. Data are representative of the mean of three independent experiments ± SD. A minimum of 40 metaphases were analyzed per experiment. The indicated p values were calculated using one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: The equilibrium dissociation constants of the WT and mutant TRF2 TRFH –MCPH1 TBM interactions were determined using a MicroCal iTC 200 calorimeter (Malvern Panalytical).

Techniques: Staining, Plasmid Preparation, Comparison, Expressing, shRNA, Labeling, Generated, Construct

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: a Representative images from three independent experiments of telomeric PNA-FISH on chromosome spreads of WT and MCPH1 Δ/Δ HCT116 cells to detect multiple telomeric signals (MTS) (arrowheads). Scale bar: 5 μm. b Mean values ± SD of the percentage of MTS visualized by PNA-FISH in the indicated cell lines treated with either DMSO or 0.25 μM aphidicolin (APH). n = three independent experiments. At least 50 metaphases were scored. One-way ANOVA followed by Tukey’s multiple comparison test. c Co-IP with anti-TRF2 antibody from lysates of synchronized U2OS cells harvested at the indicated time points. Cyclin A was used as a control for cell cycle progression. Representative blots from two independent experiments. No Ab: no antibody control; *non-specific band. See also Supplementary Fig. . d Percentage of cells showing ≥4 MCPH1/FLAG-TRF1 co-localizations in IMR-90 and HeLa cells expressing CDT1 (G 1 ) and Geminin (S/G 2 ). See also Supplementary Fig. . Mean values ± SD from two independent experiments are shown. At least 200 nuclei were scored. Two-sided Student’s t test. e SMARD analysis of telomeric DNA fibers in U2OS treated with either Scrambled or MCPH1 shRNAs. Top: scheme of the CldU (green) and IdU (red) pulse label timing. Middle: representative images of telomeric fibers (telomeric DNA depicted in blue) from two independent experiments. Bottom: quantification of either CldU- or IdU-positive telomeric fibers. Scale bar: 10 μm. f Quantification of the length of CldU and IdU tracks from a representative experiment. Blue line: median. Two-sided Mann–Whitney test. g SMARD analysis of telomeric replication forks restart in U2OS cells treated with either Scrambled or MCPH1 shRNA and reconstituted with the indicated constructs. Top: pulse labeling timing scheme with hydroxyurea (HU)-induced replication block. Bottom: Representative images from two independent experiments. The dashed line separates the CldU-labeled portion (regular replication) and the IdU-labeled portion (replication restart). Scale bar: 10 μm. h Quantification of the IdU/CldU length ratio for the fibers labeled with both halogenated nucleotides in one representative experiment. Black line: median. Kruskal–Wallis test with Dunn’s multiple comparison test.

Article Snippet: The equilibrium dissociation constants of the WT and mutant TRF2 TRFH –MCPH1 TBM interactions were determined using a MicroCal iTC 200 calorimeter (Malvern Panalytical).

Techniques: Comparison, Co-Immunoprecipitation Assay, Control, Expressing, MANN-WHITNEY, shRNA, Construct, Labeling, Blocking Assay

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: a MCPH1 (red) and γ-H2AX (green) immunostaining in the indicated cells lines treated with either DMSO or 0.25 μM APH. Representative images from either three (MCPH1 +/+ and MCPH1 Δ/Δ + vector cells) or two (MCPH1 Δ/Δ + WT MCPH1 cells) independent experiments. b Mean values ± SD of the percentage of cells with >5 MCPH1/γ-H2AX co-localizations from ( a ). n = 3 for MCPH1 +/+ and MCPH1 Δ/Δ + vector cells, n = 2 for MCPH1 Δ/Δ + WT MCPH1 cells. At least 200 nuclei were scored. One-way ANOVA followed by Tukey’s multiple comparison test. c MCPH1 (red) and SMARCAL1 (green) immunostaining in U2OS cells treated with either DMSO or 0.25 μM APH. Representative images from three independent experiments. d Mean values ± SD of the percentage of cells with >5 co-localizing MCPH1/SMARCAL1 foci from ( c ). n = 3., at least 200 cells scored. Two-sided Student’s t test. e Percentage of cells with >5 BARD1, RAD51 or p-RPA32 (S4/S8) foci co-localizing with γ-H2AX in the indicated cell lines treated with either DMSO or 0.25 μM APH. Representative images are shown in Supplementary Fig. . Data represent mean values ± SD, n = 2. At least 200 nuclei were examined. One-way ANOVA followed by Tukey’s multiple comparison test. f Representative images from two independent time course experiments to assess γ-H2AX foci (green) resolution in the indicated cell lines after release of HU block. Cells were incubated with 2 mM HU for 24 h before replacing the media and then fixed at the indicated time points. Untreated cells were used as control. g Number of γ-H2AX foci observed in 50–100 cells from each sample shown in ( f ). Data from one representative experiment. Red line: median. Kruskal–Wallis test followed by Dunn’s multiple comparison test. h , i Cell viability assay of MCPH1 +/+ and MCPH1 Δ/Δ cells treated with increasing amount of HU ( h ) or Olaparib ( i ), with or without concomitant BRCA1 depletion. Data from one of three independent experiments. Statistical significance is shown in Supplementary Table (HU) and 2 (Olaparib). Scale bars for a , c , f : 5 μm.

Article Snippet: The equilibrium dissociation constants of the WT and mutant TRF2 TRFH –MCPH1 TBM interactions were determined using a MicroCal iTC 200 calorimeter (Malvern Panalytical).

Techniques: Immunostaining, Plasmid Preparation, Comparison, Blocking Assay, Incubation, Control, Viability Assay

Journal: Nature Communications

Article Title: Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly

doi: 10.1038/s41467-020-19674-0

Figure Lengend Snippet: MCPH1 interacts with TRF2 when S333 is de-phosphorylated, while this interaction is disrupted upon S333 phosphorylation. In unperturbed conditions, MCPH1 exists in equilibrium between the phosphorylated and the de-phosphorylated forms and its telomeric localization increases in S phase. Removal of POT1 through the overexpression of TPP1 ΔRD increases MCPH1 localization to telomeres that is dependent upon the interaction with both TRF2 and γ-H2AX. MCPH1 binding to TRF2 promotes the recruitment of HDR and end resection factors to initiate HDR at telomeres lacking POT1-TPP1. Cells lacking MCPH1 or expressing MCPH1 S333D display reduced recruitment of HDR factors and reduced T-SCEs, suggestive of HDR defects. Similarly, induction of replication stress at telomeres increases MCPH1 telomeric localization, promoted by its interaction with TRF2. In the absence of MCPH1 or upon overexpression of the phosphomimetic mutant MCPH1 S333D , replication forks stalling at telomeres increases and telomere replication is impaired, resulting in telomere fragility. These observations suggest that telomeric localization of MCPH1 is required for proper telomere replication by promoting stalled replication fork restart.

Article Snippet: The equilibrium dissociation constants of the WT and mutant TRF2 TRFH –MCPH1 TBM interactions were determined using a MicroCal iTC 200 calorimeter (Malvern Panalytical).

Techniques: Over Expression, Binding Assay, Expressing, Mutagenesis

Journal: Journal of neurochemistry

Article Title: TRF2 dysfunction elicits DNA damage responses associated with senescence in proliferating neural cells and differentiation of neurons.

doi: 10.1111/j.1471-4159.2006.03779.x

Figure Lengend Snippet: Fig. 1 Expression of dominant negative telomeric repeat binding factor 2 (DN-TRF2) displaces telomeric repeat binding factor 2 (TRF2) from telomeres in neural cells. (a) Structure of full-length and DN-TRF2 constructs. The cDNA sequence of the cloned rat TRF2 (rTRF2) in the dimerization domain region was confirmed by sequencing and is highly homologous with human TRF2 (hTRF2). (b) A representative immunoblot illustrating the overexpression of wild- type TRF2 (WT-TRF2) and DN-TRF2 in SH-SY5Y cells 24 h after transfection. The blot was probed with a TRF-2 antibody and shows relative levels of WT-TRF2 (66 kDa, single arrow) and DN-TRF2 (50 kDa, double arrow). (c) DN-TRF2 displaces WT-TRF2 from te- lomeres. SH-SY5Y human neuroblastoma cells were transfected with YFP-TRF1 to label telomeres, in combination with WT-TRF2 or DN- TRF2. Cells were then immunostained 24 h after transfection with a TRF2 antibody. Confocal images show YFP-TRF1 (green) and TRF2 (red) associated fluorescence; these images are representative of

Article Snippet: After pre-incubation with blocking buffer (2% non-fat powdered milk, 2% normal serum and 0.2% Triton X-100 in phosphate-buffered saline) for 1 h, cells were incubated with a primary antibody in blocking buffer overnight at 4 C. The primary antibodies used were: anti-TRF2 monoclonal antibody (Imgenex, San Diego, CA, USA); anti-cH2AX monoclonal and polyclonal antibodies (Upstate Biotechnology, Lake Placid, NY, USA); ATM 1918 monoclonal antibody (Upstate Biotechnology) and ATM S/TQ substrate polyclonal antibody (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Dominant Negative Mutation, Binding Assay, Construct, Sequencing, Clone Assay, Western Blot, Over Expression, Transfection

Journal: Journal of neurochemistry

Article Title: TRF2 dysfunction elicits DNA damage responses associated with senescence in proliferating neural cells and differentiation of neurons.

doi: 10.1111/j.1471-4159.2006.03779.x

Figure Lengend Snippet: Fig. 3 Dysfunctional telomeric repeat binding factor 2 (TRF2) induces divergent ATM-mediated DNA damage responses in hippocampal neurons and mitotic neural cells. (a and b) Telomere-directed ATM and cH2AX activations were triggered by the expression of dominant negative TRF2 (DN-TRF2) in hippocampal neurons and SH-SY5Y cells. (a) Hippocampal neurons were transfected either with eGFP vector or with DN-TRF2 in combination with the IRES-GFP reporter gene, and were then immunostained with anti-cH2AX polyclonal (green) and anti-ATM1981 (red) monoclonal antibodies. Note that in cells expressing DN-TRF2, many cH2AX foci co-localize with phos- phorylated ATM1981 (single arrows in inset point to dual stained foci). (b) SH-SY5Y cells were co-transfected with YFP-TRF1 and wild-type TRF2 (WT-TRF2) or with YFP-TRF1 and DN-TRF2. The cells were then immunostained with anti-cH2AX polyclonal (green) and anti- ATM1981 (red) monoclonal antibodies. Note that in cells expressing

Article Snippet: After pre-incubation with blocking buffer (2% non-fat powdered milk, 2% normal serum and 0.2% Triton X-100 in phosphate-buffered saline) for 1 h, cells were incubated with a primary antibody in blocking buffer overnight at 4 C. The primary antibodies used were: anti-TRF2 monoclonal antibody (Imgenex, San Diego, CA, USA); anti-cH2AX monoclonal and polyclonal antibodies (Upstate Biotechnology, Lake Placid, NY, USA); ATM 1918 monoclonal antibody (Upstate Biotechnology) and ATM S/TQ substrate polyclonal antibody (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Binding Assay, Expressing, Dominant Negative Mutation, Transfection, Plasmid Preparation, Bioprocessing, Staining

Journal: Journal of neurochemistry

Article Title: TRF2 dysfunction elicits DNA damage responses associated with senescence in proliferating neural cells and differentiation of neurons.

doi: 10.1111/j.1471-4159.2006.03779.x

Figure Lengend Snippet: Fig. 4 Inhibition of telomeric repeat binding factor 2 (TRF2) function induces senescence and p53 expression in proliferating neural cells, but not in neurons. (a) Dominant negative TRF2 (DN-TRF2) induced senescence in SH-SY5Y cells. The cells were stably transfected with either vector or DN-TRF2 for 15 days. The growth rate of the cells was then quantified by counting cells in defined microscope fields during the next 7 days. The cells were then treated with 1 lM BrdU for 1 h, and then fixed and stained with an anti-BrdU antibody or SA-b- galactosidase (SA-b-Gal) reagent. Compared with vector control (i), expression of DN-TRF2 (ii) resulted in a significant increase of b-galactosidase levels (v; **p < 0.001), a significant decrease in the number of BrdU-labeled cells (v; **p < 0.001) and a significant decrease in the cell growth rate (vi; p < 0.001). Values are the mean and SEM of determinations made in at least six different cultures. (b) DN-TRF2 induced senescence in astrocytes. Astrocytes were infected with adenoviral vectors containing either GFP or DN-TRF2 (50 pfu/ cell). Compared with controls (i), expression of DN-TRF2 (ii) resulted in a significant increase in the number of b-galactosidase positive

Article Snippet: After pre-incubation with blocking buffer (2% non-fat powdered milk, 2% normal serum and 0.2% Triton X-100 in phosphate-buffered saline) for 1 h, cells were incubated with a primary antibody in blocking buffer overnight at 4 C. The primary antibodies used were: anti-TRF2 monoclonal antibody (Imgenex, San Diego, CA, USA); anti-cH2AX monoclonal and polyclonal antibodies (Upstate Biotechnology, Lake Placid, NY, USA); ATM 1918 monoclonal antibody (Upstate Biotechnology) and ATM S/TQ substrate polyclonal antibody (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Inhibition, Binding Assay, Expressing, Dominant Negative Mutation, Stable Transfection, Transfection, Plasmid Preparation, Microscopy, Staining, Control, Labeling, Infection

Journal: Circulation

Article Title: Statins Enhance Migratory Capacity by Upregulation of the Telomere Repeat-Binding Factor TRF2 in Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000142866.50300.eb

Figure Lengend Snippet: Figure 2. Induction of TRF2 protein expression by statins. A, Mononuclear cells were isolated from healthy donors, and whole protein extracts were generated on day 3 from EPCs. Represen- tative immunoblots from 3 different donors are shown, using Abs against TRF2 (H-300), Chk2, and -actin. Cells were incu- bated either with 0.1% DMSO (Co), atorvastatin (At; 0.1 mol/L), or mevastatin (Me; 1.0 mol/L). B, Semiquantitative analysis of TRF2 immunoblot (A) by densitometry. Control value is defined as 100%. All values are presented as meanSEM. §P0.01, #P0.001 vs control. C, Mononuclear cells were isolated from healthy donors, and whole protein extracts were generated on

Article Snippet: The following primary Abs were used: -actin mouse monoclonal Ab (Sigma A5441; 1:8000), TRF2 H-300 rabbit polyclonal Ab (Santa Cruz sc-9143; 1:250), TRF2 goat polyclonal Ab (Santa Cruz sc-8528; 1:250), Checkpoint-kinase 2 (Chk2) mouse monoclonal Ab (Santa Cruz sc-17747; 1:250), green fluorescent protein (GFP) rabbit polyclonal Ab (Invitrogen 46-0092; 1:3000), and c-myc 9B11 mouse monoclonal Ab (Cell Signaling 2276; 1:500).

Techniques: Expressing, Isolation, Generated, Western Blot, Control

Journal: Circulation

Article Title: Statins Enhance Migratory Capacity by Upregulation of the Telomere Repeat-Binding Factor TRF2 in Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000142866.50300.eb

Figure Lengend Snippet: Figure 3. Regulation of Chk2 in cultured EPCs. A, Immunoblot showing upregulation of Chk2 in EPCs, cultured for 3 days with- out addition of atorvastatin (AT; 0.1 mol/L). Freshly isolated mononuclear cells were used as a control (Co) for day 0. A rep- resentative blot from 2 donors is shown. B, Semiquantitative analysis of Chk2 immunoblot (Figure 2A) by densitometry. Con- trol value is defined as 100%. All values are presented as meanSEM. #P0.001 vs control. C, Overexpression of wild- type TRF2 in freshly isolated mononuclear cells abrogates induction of Chk2 in day 3 EPCs. Increasing concentrations of Antennapedia peptide (Antp) were used during adenoviral infec- tion as indicated. A representative blot from 2 experiments is shown.

Article Snippet: The following primary Abs were used: -actin mouse monoclonal Ab (Sigma A5441; 1:8000), TRF2 H-300 rabbit polyclonal Ab (Santa Cruz sc-9143; 1:250), TRF2 goat polyclonal Ab (Santa Cruz sc-8528; 1:250), Checkpoint-kinase 2 (Chk2) mouse monoclonal Ab (Santa Cruz sc-17747; 1:250), green fluorescent protein (GFP) rabbit polyclonal Ab (Invitrogen 46-0092; 1:3000), and c-myc 9B11 mouse monoclonal Ab (Cell Signaling 2276; 1:500).

Techniques: Cell Culture, Western Blot, Isolation, Control, Over Expression

Journal: Circulation

Article Title: Statins Enhance Migratory Capacity by Upregulation of the Telomere Repeat-Binding Factor TRF2 in Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000142866.50300.eb

Figure Lengend Snippet: Figure 4. Statin-enhanced migration in EPCs depends on TRF2 function. A, Viral vector. Dominant-negative TRF2 (TRF2BM) tagged with myc epitope lacks Myb domain and N-terminal basic domain, coexpressing enhanced GFP.33 Western blots confirming expression of exogenous proteins in EPCs. Dominant negative TRF2 is detected with H-300 Ab (raised against amino acids 49 to 300; Santa Cruz Biotechnology) but not C-16 (raised against C terminus, data not shown). B and C, Freshly isolated mononuclear cells were infected with adenoviral vectors Ad-GFP (GFP) and Ad-TRF2dn (dn), respectively. Atorvastatin (AT; 0.1 mol/L) was added to media where indicated. Migratory activity of EPCs was determined on day 3 after adenoviral infec- tion either without (B) or with (C) addition of 100 ng/mL SDF into lower chamber. Number of Dil-positive cells/HPF was counted. Data are meanSEM. Three parallel experiments with n3 HPFs each were included in analysis. Levels of significance between pairs of groups in B and C are indicated by following symbols: *P0.05; §P0.01; #P0.001.

Article Snippet: The following primary Abs were used: -actin mouse monoclonal Ab (Sigma A5441; 1:8000), TRF2 H-300 rabbit polyclonal Ab (Santa Cruz sc-9143; 1:250), TRF2 goat polyclonal Ab (Santa Cruz sc-8528; 1:250), Checkpoint-kinase 2 (Chk2) mouse monoclonal Ab (Santa Cruz sc-17747; 1:250), green fluorescent protein (GFP) rabbit polyclonal Ab (Invitrogen 46-0092; 1:3000), and c-myc 9B11 mouse monoclonal Ab (Cell Signaling 2276; 1:500).

Techniques: Migration, Plasmid Preparation, Dominant Negative Mutation, Western Blot, Expressing, Isolation, Infection, Activity Assay