Journal: PLoS Genetics

Article Title: ATM Release at Resected Double-Strand Breaks Provides Heterochromatin Reconstitution to Facilitate Homologous Recombination

doi: 10.1371/journal.pgen.1003667

Figure Lengend Snippet: ( A ) A549 tumor cells treated with BRCA2 siRNA were irradiated with 1 Gy (0.5 h) or 2 Gy (8 h) and immunostained with the indicated antibodies. Using EdU and cell cycle markers to distinguish G1- from G2-phase cells , focal intensities of pATM were measured using ImageJ software (see ). BRCA2 siRNA was used in this analysis to accumulate resected DSBs. ( B ) 2BN hTert (XLF-deficient) human fibroblasts were analyzed 2 h post IR with 1 Gy. Cells were stained against γH2AX and RAD51 or pATM and RAD51, and γH2AX or pATM focal intensities were measured at RAD51-foci-positive or RAD51-foci-negative foci. XLF-deficient cells were used in this analysis to prevent repair of EC DSBs during the time needed for resection of HC DSBs. ( C ) 82-6 hTert (wt) and F02-98 hTert (ATR-deficient) human fibroblasts were stained against γH2AX and RAD51 at 2 h post 1 Gy, and γH2AX focal intensities were measured as in (B). ( D ) 2BN hTert (XLF-deficient) human fibroblasts were stained against γH2AX and RAD51 at 2 h post 1 Gy, and γH2AX focal intensities were measured as in (B). Since both DNA-PK and ATM can phosphorylate γH2AX, cells were treated with DNA-PK inhibitor under all conditions. Inhibitors were added 1 h post IR, a time sufficient to allow for ATM-dependent resection and RAD51 loading (see ). In (A–D), at least 300 foci from 3 independent experiments were analyzed for each point. Box plots were used with a maximum whisker-length of 1.5-fold the inter-quartile range; the lower and upper “x” indicates the 1% or 99% margin of the data range, respectively.

Article Snippet: Antibodies used were: mouse-α-γH2AX at 1∶2000 (Millipore); rabbit-α-γH2AX at 1∶2000 (Abcam), mouse-α-pATM at 1∶1000 (Biomol), rabbit-α-RAD51 at 1∶15000 (Abcam), mouse-α-RPA at 1∶2000 (Neomarkers) and mouse-α-GFP at 1∶200 (Roche).

Techniques: Irradiation, Software, Staining, Whisker Assay

Journal: PLoS Genetics

Article Title: ATM Release at Resected Double-Strand Breaks Provides Heterochromatin Reconstitution to Facilitate Homologous Recombination

doi: 10.1371/journal.pgen.1003667

Figure Lengend Snippet: ( A ) G1-synchronized and G2-enriched A549 tumor cells were irradiated with 10 Gy and harvested at indicated time points. Fractionated chromatin (chromatin) was immunoblotted (left panel), and pATM, pKAP-1 and γH2AX levels of the chromatin fraction from the same blot were quantified using ImageJ. The right panels show the ratio of pATM or pKAP-1 relative to γH2AX for G1 and G2 cells at various time points. The data for G2 was normalized to G1 which was set to 100% (mean ± SEM from ≥2 experiments). ( B ) G2-synchronized HeLa tumor cells were irradiated with 30 Gy, harvested at the indicated time points, immunoprecipitated (IP) with γH2AX antibody and analyzed by immunoblotting. In G2 cells, pATM is co-immuno-precipitated with γH2AX at 30 min but not at 8 h post IR. The depicted FACs distributions in panels (A) and (B) represent the cell populations at the time of irradiation. How these populations change during repair incubation is shown in . ( C ) HeLa tumor cells were treated with siRNA, synchronized in G2, and whole cell extracts were analyzed by immunoblotting 4 h post 10 Gy. pKAP-1 is detected in unsynchronized but not in G2-synchronized cells unless either CtIP or BLM is depleted. Depletion of CtIP or BLM did not affect the cell cycle distribution (see ). ( D ) G2-synchronized HeLa tumor cells were irradiated with 30 Gy, harvested at the indicated time points, immunoprecipitated (IP) with γH2AX antibody and analyzed by immunoblotting. The level of KAP-1 co-immuno-precipitated with γH2AX increases with increasing repair time post IR. KAP-1 is substantially phosphorylated at early but not at later times.

Article Snippet: Antibodies used were: mouse-α-γH2AX at 1∶2000 (Millipore); rabbit-α-γH2AX at 1∶2000 (Abcam), mouse-α-pATM at 1∶1000 (Biomol), rabbit-α-RAD51 at 1∶15000 (Abcam), mouse-α-RPA at 1∶2000 (Neomarkers) and mouse-α-GFP at 1∶200 (Roche).

Techniques: Irradiation, Immunoprecipitation, Western Blot, Incubation

Journal: EMBO Molecular Medicine

Article Title: DNA-dependent protein kinase catalytic subunit mediates T-cell loss in rheumatoid arthritis

doi: 10.1002/emmm.201000096

Figure Lengend Snippet: CD4 + CD45RO − T cells were isolated from the peripheral blood of RA patients and controls. Cells were maintained in culture without mitogenic stimulation for 72 h. T cells undergoing apoptosis were identified by Annexin V and 7-AAD staining in patient-derived and control samples. Data are given as mean ± SEM from six patients and six controls. Representative data from one patient and one control collected after 72 h are shown. Cells were maintained in culture with IL-2 (20 U/ml), IL-7 (20 ng/ml), IL-15 (10 ng/ml) or a mixture of the three (Mix) for 72 h. T cells undergoing apoptosis were identified by PI staining. Data are given as mean ± SEM from seven patients and five controls. DNA breaks were quantified by comet assay after 72 h. Data are given as mean ± SEM of seven patients and six controls with a minimum of 90 individual cells analyzed in each sample. Flow cytometry analysis of 8-oxoguanine levels in control (dashed line) and RA T cells (solid line). Unstained control is shown as light grey. The level of 8-oxoguanine in five RA and five control samples is given as mean fluorescence intensity (MFI) of FITC-8-oxoguanine ± SEM. 53BP1 foci were determined by immunofluorescence staining and confocal laser microscopy after 72 h. Bar, 20 µM. The levels of 53BP1 foci in three RA and three control samples is given as total 53BP1 intensity per nucleus ± SEM and 53BP1 foci per nucleus ± SEM.

Article Snippet: After overnight incubation, 10 7 cells/ml were stained with fluorescein isothiocyanate (FITC)-pATM (Rockland Immunochemicals, Gilbertsville, PA), Alexa Fluor 488-pp53, FITC-Bcl-2, FITC-pJNK, Alexa Fluor 488-Bim, PE-Bmf (Cell Signalling Technology, Danvers, MA), Alexa Fluro 488-Ku70 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies, or FITC-conjugated 8-oxoguanine probe (OxyDNA Assay Kit; Calbiochem).

Techniques: Isolation, Staining, Derivative Assay, Control, Single Cell Gel Electrophoresis, Flow Cytometry, Fluorescence, Immunofluorescence, Microscopy

Journal: EMBO Molecular Medicine

Article Title: DNA-dependent protein kinase catalytic subunit mediates T-cell loss in rheumatoid arthritis

doi: 10.1002/emmm.201000096

Figure Lengend Snippet: CD4 + CD45RO − T cells were isolated from six controls and six RA patients. Cells were maintained in culture without stimulation and collected after 72 h. T cells undergoing apoptosis were stained with PE-caspase-3 and phosphorylated ATM was detected by FITC-Phosp-ATM. Representative cytometric data from one patient and one control are shown. Expression levels of pATM were quantified by flow cytometry in five RA and five control samples and are given as mean MFI of FITC-pATM ± SEM. Expression levels of pp53 were quantified by flow cytometry in six RA and six control samples and are given as mean MFI of Alexa Fluor 488-pp53 ± SEM.

Article Snippet: After overnight incubation, 10 7 cells/ml were stained with fluorescein isothiocyanate (FITC)-pATM (Rockland Immunochemicals, Gilbertsville, PA), Alexa Fluor 488-pp53, FITC-Bcl-2, FITC-pJNK, Alexa Fluor 488-Bim, PE-Bmf (Cell Signalling Technology, Danvers, MA), Alexa Fluro 488-Ku70 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies, or FITC-conjugated 8-oxoguanine probe (OxyDNA Assay Kit; Calbiochem).

Techniques: Isolation, Staining, Control, Expressing, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: DNA-dependent protein kinase catalytic subunit mediates T-cell loss in rheumatoid arthritis

doi: 10.1002/emmm.201000096

Figure Lengend Snippet: CD4 + CD45RO − T cells were isolated and cultured in the presence of the indicated enzyme inhibitors for 24 h. T-cell apoptosis was quantified through flow cytometry staining for PI. Cells were maintained in culture without stimulation for 48 h and then were treated with the JNK inhibitor. PI-positive T cells were measured after 24 h of treatment with the JNK inhibitor II (5 or 10 µM) or vehicle. Frequencies of apoptotic T cells from six controls and seven RA patients are presented as mean ± SEM. Representative flow cytometry results from one control and one patient. Flow cytometry analysis of phosphorylated JNK levels in control and RA T cells after culturing without mitogenic stimulation for 72 h. Representative data from one patient and one control are presented. Expression of pJNK protein in n = 6 RA and n = 6 control samples at 72 h is given as MFI of FITC-pJNK. Flow cytometry analysis of phosphorylated JNK levels in freshly isolated control and RA naïve CD4 T cells. Expression of pJNK protein in n = 7 RA and n = 7 control samples at day 0 is given as MFI of FITC-pJNK. Quantification of JNK and pJNK protein expression at 72 h by Western blotting. Relative expression levels of pJNK were quantified by measuring band intensities adjusted by total JNK in six RA patients and six control donors. Data are presented as mean ± SEM. CD4 + CD45RO − T cells were purified from RA donors and transfected with control or DNA-PKcs-specific siRNA oligonucleotides by nucleofection. Twenty-four hours after transfection, DNA-PKcs and pJNK protein levels were detected by Western blotting. A representative blot from three independent experiments is shown.

Article Snippet: After overnight incubation, 10 7 cells/ml were stained with fluorescein isothiocyanate (FITC)-pATM (Rockland Immunochemicals, Gilbertsville, PA), Alexa Fluor 488-pp53, FITC-Bcl-2, FITC-pJNK, Alexa Fluor 488-Bim, PE-Bmf (Cell Signalling Technology, Danvers, MA), Alexa Fluro 488-Ku70 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies, or FITC-conjugated 8-oxoguanine probe (OxyDNA Assay Kit; Calbiochem).

Techniques: Isolation, Cell Culture, Flow Cytometry, Staining, Control, Expressing, Western Blot, Purification, Transfection

Journal: EMBO Molecular Medicine

Article Title: DNA-dependent protein kinase catalytic subunit mediates T-cell loss in rheumatoid arthritis

doi: 10.1002/emmm.201000096

Figure Lengend Snippet: CD4 + CD45RO − T cells were isolated from the peripheral blood of controls and RA patients. Cells were analyzed immediately after isolation or maintained in culture without mitogenic stimulation for 72 h. DNA-PKcs or JNK inhibitors were added for the final 24 h of culture. Puma, Noxa, Bim and Bmf transcript levels in freshly isolated cells were quantified by qPCR. Data are presented as mean ± SEM. Flow cytometry analysis of Bim levels in control and RA T cells after 72 h of culture. Expression of Bim protein in n = 5 RA and n = 5 controls is given as MFI of Alexa Fluor 488 Bim. Flow cytometry analysis of Bmf levels in control and RA T cells after 72 h of culture. Expression of Bmf protein in n = 6 RA and n = 4 controls is given as MFI of PE Bmf. Bcl-2 protein levels were analyzed by flow cytometry after 72 h of culture. Expression of Bcl-2 protein in n = 6 RA and n = 6 control samples is given as MFI of FITC Bcl-2. Bim protein levels in the absence or presence of the DNA-PKcs inhibitor IC86621 (100 nM) were analyzed by flow cytometry in samples from four RA patients. Results are presented as MFI ± SEM. Bim levels in the absence or presence of the JNK inhibitor II (10 µM) were analyzed in naïve T cells from six RA patients. Results are presented as MFI ± SEM. Flow cytometry analysis of Bmf protein expression in T cells from three RA patients in the absence or presence of the DNA-PKcs inhibitor II. Results are presented as MFI ± SEM. Flow cytometry analysis of Bmf protein expression in T cells from three RA patients in the absence or presence of the JNK inhibitor II. Results are presented as MFI ± SEM.

Article Snippet: After overnight incubation, 10 7 cells/ml were stained with fluorescein isothiocyanate (FITC)-pATM (Rockland Immunochemicals, Gilbertsville, PA), Alexa Fluor 488-pp53, FITC-Bcl-2, FITC-pJNK, Alexa Fluor 488-Bim, PE-Bmf (Cell Signalling Technology, Danvers, MA), Alexa Fluro 488-Ku70 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies, or FITC-conjugated 8-oxoguanine probe (OxyDNA Assay Kit; Calbiochem).

Techniques: Isolation, Flow Cytometry, Control, Expressing