Computational protocol: DNA ligase III acts as a DNA strand break sensor in the cellular orchestration of DNA strand break repair

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[…] Cells were plated on 35-mm glass bottom dishes at ∼70–80% confluency. For immunofluorescence cells were fixed and permeabilized using ice cold methanol:acetone (1:1) mixture for 20 min. Then cells were rehydrated with PBS at room temperature for 15 min. For 8-oxo-dG staining, cells were treated with 2N HCl for 10 min at 37ºC, then washed with PBS (three 5-min washes) prior to incubation with primary antibody. Subsequently cells were blocked with 5% bovine serum albumin for 1 h and incubated with primary antibodies for 1 h at room temperature or overnight at 4ºC. After incubation, cells were washed once with 0.1% Triton-X-100 in PBS, then twice in PBS. Finally, cells were incubated with appropriate secondary antibodies for 30–45 min at room temperature, and then washed as described above. In the final wash, 4′,6-diamidino-2-phenylindole (DAPI) was added for nuclear DNA staining and left for 15 min then washed with PBS. Primary antibodies used included mouse monoclonal anti-8-oxo-dG (cat. no. 4354-MC-050, Trevigen, Gaithersburg, MD, USA) (1:250 dilution), mouse monoclonal anti-γH2AX (cat. no. 05–636, Millipore, Billerica, MA, USA) (1:5000 dilution), mouse monoclonal anti-53BP1 (Millipore) (1:400 dilution), mouse monoclonal anti-XRCC1 (ab1838–250, Abcam, Cambridge, MA, USA) (1:500 dilution), rabbit polyclonal anti-DNA Ligase 3 (GTX103197, GeneTex, Irvine, CA, USA) (1:250 dilution) and mouse monoclonal anti-poly(ADP-ribose) (ab14459, Abcam) (1:5000 dilution). Secondary antibodies used were Alexa Fluor 555 goat anti-rabbit (A-21430, Invitrogen, Burlington, ON, USA) (1:200 dilution) and Alexa Fluor 488 goat anti-mouse (A-11017, Invitrogen) (1:200) conjugated antibodies. For image acquisition, fixed and stained cells were placed on the stage of a Zeiss confocal LSM 710 microscope. Images were acquired using either 40X or 63X objectives as 12 bit grayscale images, and then exported as Tiff 16 bit grayscale images for processing using ImageJ software. For immunofluorescence experiments, three independent experiments were carried out. In each experiment 15–20 cell images were acquired and analyzed. [...] For two-photon laser micro-irradiation, cells were grown on 35-mm glass bottom dishes. Before laser micro-irradiation, cells were incubated with Hoechst 33258 (Sigma, cat. no. 94403) to a final concentration of 1 μg/ml for 20 min and then fed with fresh growth medium for 10 min. Where indicated, cells were incubated with either 1- or 2-μM AG14361 (IC50 = 29 nM, Selleckchem, cat. no. S2178), or with 2.5- and 10-μM PJ-34 (IC50 = 20 nM, Enzo Life Sciences, cat. no. ALX-270–289), for 1–2 h prior to micro-irradiation. Subsequently, cells were placed on a 37ºC-heated stage of a Zeiss LSM510 NLO laser-scanning confocal microscope. Micro-irradiation was carried out using a near-infrared titanium sapphire laser. To introduce damage within nuclei of individual cells, a 1.2-μm-wide region was pre-defined and subsequently micro-irradiated with 10 iterations of a 750-nm (50 mW) laser line at 10% power using a Plan-Neofluar 40X/1.3 NA oil immersion objective. For immunofluorescence of endogenous proteins and protein modifications, cells were fixed right after laser micro-irradiation and counterstained with antibodies of interest. For time lapse experiments of mRFP-tagged proteins, the fluorescent signal was recorded using excitation with a 543-nm He–Ne laser and a 559–634-nm band-pass emission filter. Similarly, for mGFP- and EGFP-tagged proteins, the signal was recorded after excitation with a 488-nm argon laser and a 515–540-nm band-pass emission filter. Cells with low to medium expression levels of fluorescent proteins were selected and accumulation of fluorescently tagged protein in micro-irradiated areas was quantified and compared to that in unirradiated regions. After background subtraction as previously described (), the intensity was normalized so that the total cell intensity remained constant throughout the experiment. This process compensates for photobleaching during acquisition (). Images were then realigned using ImageJ software and fluorescence signals of the exported Tiff images were subsequently quantified using Metamorph software 6.0 (Molecular Devices). Plotted results of recruitment kinetics represent averages of three independent experiments. For each experiment 10–12 cells were analyzed (total 30–36 cells).For the 405-nm laser micro-irradiation, we applied the same settings as described by Dinant et al. (). Briefly, cells transiently expressing a fluorescent protein-tagged protein of interest were pre-sensitized with Hoechst dye for 20 min, at a final concentration of 0.5 μg/ml, and then the media was replaced prior to micro-irradiation. Cells were placed on a 37ºC-heated stage of a Zeiss LSM710 NLO laser-scanning confocal microscope. To introduce damage within nuclei of individual cells, a 1.2-μm-wide region was pre-defined and subsequently micro-irradiated with 30 iterations of a 405-nm (30 mW) laser line at 60% power using a Plan-Neofluar 63X/1.3 NA oil immersion objective. For time lapse experiments of EGFP/mGFP-tagged proteins, the signal was recorded after excitation with a 488-nm argon laser and a 515–540-nm band-pass filter. Cells with low to medium expression levels of fluorescent proteins were selected and analyzed. For quantification, analyses were performed as previously described (). […]

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