Computational protocol: PARP3 is a promoter of chromosomal rearrangements and limits G4 DNA

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Protocol publication

[…] At the indicated times after irradiation, cells were pre-extracted with 25 mM HEPES, pH 7.4, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 300 mM sucrose and 0.5% Triton X-100 for 5 min on ice. Cells were fixed with 4% paraformaldehyde for 12 min at room temperature, washed twice with PBS with 1% FBS for blocking and co-stained for 16 h at 4 °C with γH2AX S139 (20E3, Cell Signaling) and RPA32 (A300-244A, Bethyl Labs) at dilutions of 1:500, mouse anti-DNA G quadruplex (G4), (1H6, EMD Millipore; dilution of 1:500), rabbit anti-BLM (A300-110, Bethyl Labs; dilution of 1:1,000), and rabbit anti-53BP1 (A300-272A, Bethyl Labs; dilution of 1:1,000). Cells were washed with PBS containing 1% FBS and stained for 2 h in the dark at room temperature with Alexa Fluor 594 goat anti-rabbit (eBioscience) and Alexa Fluor 488 goat anti-mouse (eBioscience) at dilutions of 1:1,000. Slides were prepared with mounting media with 4,6-diamidino-2-phenylindole (DAPI; Vectashield). Confocal immunofluorescence images were collected at 405, 488 and 561 nm using a Yokogawa CSU-X1 spinning disk confocal (Andor Technology) mounted on a Nikon Ti-E inverted microscope (Nikon Instruments, Melville, NY). Images were acquired using a × 40 Plan Apo NA 1.0 oil objective with an Andor iXon 897 EMCCD camera. Acquisition parameters, shutters and filter positions were controlled using the Andor iQ software. Confocal microscopy images for this study were acquired in the Confocal and Light Microscopy Core facility at the Dana-Farber Cancer Institute. Additional images were acquired using a Zeiss AxioImager Z1 microscope equipped with an Axiocam MRc Rev.3 colour digital camera and Plan APO × 63/1.4 oil M27 lens (magnification × 63). Acquisition software and image processing used the Zeiss AxioVision software package (Zeiss Imaging). Images were analysed with the US National Institutes of Health Image J FIJI program (http://rsbweb.nih.gov.ezp-prod1.hul.harvard.edu/ij/index.html). Specifically, ImageJ FIJI was used to quantify foci in immunofluorescence experiments. Images from the vehicle-treated condition were used to set an appropriate noise tolerance for each antibody stain. The same noise threshold was used for all images in a given experiment (noise tolerances were as follows: 10 for γH2AX (JBW301), 10 for G4 DNA (1H6) and 5 for 53BP1 (Bethyl)). The ‘Find Maxima’ tool was used to count foci for the indicated number of nuclei. Just another co-localization plugin (Jacop) was used to calculate degree of co-localization between foci. […]

Pipeline specifications

Software tools ImageJ, JACoP
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens, Mus musculus
Diseases Neoplasms
Chemicals Poly Adenosine Diphosphate Ribose, Zinc