Bu1 75 icr1

The following primary antibodies were used: anti–E-cadherin (1:100, cat. Asp155-Ile707; R&D Systems, Minneapolis, MN), anti-BrdU (1:100, cat. BU1/75 [ICR1]; NOVUS Biologicals, Centennial, CO), anti–ZO-1 (1:100, cat. 61-7300; Thermo Fisher Scientific, Waltham, MA), anti–claudin-1 (1:200, cat. ab15098; Abcam, Waltham, MA), anti–claudin-2 (1:200, cat. ab53032; Abcam), and anti–occludin-1 (1:50, cat. 40-4700; Thermo Fisher Scientific). The following secondary antibodies were used: anti-goat (1:500, A11058; Life Technology, Carlsbad, CA), anti-rat (1:500, cat. ab150160; Abcam), and anti-rabbit (1:500, cat. A-11037; Thermo Fisher Scientific). 4’,6-diamidino-2-phenylindole (cat. 0100-20; Southern Biotech, Birmingham, AL) was used for counterstaining for immunofluorescence and FISH. Fluorophore-conjugated antibodies against CD4 (GK1.5, RM4-5), CD45 (30-F11), CD90.2 (53-2.1), CD127 (A7R34), IFN-gamma (XMG1.2), IL17A (TC11-18H10.1), and corresponding isotype controls were from BioLegend (San Diego, CA), and antibodies against FoxP3 (FJK-16s), IL22 (1H8PWSR), RORγt (B2D), Natural Killer Cell P46-Related Protein (29A1.4), hematopoietic lineage cocktail (88-7772-72), IL10 (JES5-16E3), and corresponding isotype controls were from Thermo Fisher Scientific.

The following reagents were used: Doxycycline (Sigma-Aldrich, D9891), MRE11 inhibitor [5-(4-hydroxybenzylidene)-2-iminothiazolidin-4-one, (Sigma-Aldrich, M9948)], IdU (Sigma-Aldrich, I7125), CldU (Sigma-Aldrich, C6891) and Histone deacetylase inhibitor, suberoylanilide hydroxamic acid (Tocris Biosciences, 4652). Mouse monoclonal anti-γH2AX (JBW301; EMD Millipore, 07-164), rabbit polyclonal anti-RAD51 (H-92; Santa Cruz, sc-8349), rabbit polyclonal anti-STING (Novus Biologicals, 2-24683), anti-phospho-STING (Ser366; Cell Signaling, 85735), rabbit monoclonal anti-phospho-TBK1 (Ser172; D52C2; Cell Signaling, 5483), rabbit polyclonal anti-phospho-STAT3 (Tyr705; Cell Signaling, 9131), rabbit monoclonal anti-CASPASE-3 (8G10; Cell Signaling, 9665), mouse monoclonal anti-PARP1 (F2; Santa Cruz, 8007), mouse monoclonal anti-γ-Tubulin (GTU-88; Sigma-Aldrich, T6557), mouse monoclonal anti-BrdU (B44; BD Biosciences, 347580) and rat monoclonal anti-BrdU (BU1/75-ICR1; Novus Biologicals, NB500-169) antibodies. Fluorescent conjugated secondary antibodies Alexa488, Alexa555, Alexa633, were purchased from Molecular Probes (Invitrogen).

Cells were pulse-labeled with 25 μM CldU (Sigma-Aldrich, C6891mg) and 250 μM IdU (Sigma-Aldrich, I7125-5g) at indicated times, with or without treatment, as reported in the experimental schemes. DNA fibers were prepared as previously reported (27 (link)). For the immunodetection of labeled tracks, the following primary antibodies were used: anti-CldU [rat monoclonal anti–5-bromo-2′-deoxyuridine (BrdU)/CldU; BU1/75 ICR1, Novus, 1:100] and anti-IdU (mouse monoclonal anti-BrdU/IdU; clone B44, Becton Dickinson, 1:50). The secondary antibodies were goat anti-mouse Alexa Fluor 594 (Abcam, ab150116, 1:200) or goat anti-rat Alexa Fluor 488 (Abcam, ab150157, 1:200). The incubation with antibodies was accomplished in a humidified chamber for 1 hour at room temperature. Images were acquired randomly from fields with untangled fibers using a Nikon Eclipse 80i fluorescence microscope, equipped with a video confocal system. The length of labeled tracks was measured using Fiji software, and values were converted into kilobases using the conversion factor of 1 μm = 2.59 kb. A minimum of 100 individual fibers was analyzed for each experiment, and the mean of at least three independent experiments is presented. Statistical analysis was performed using GraphPad Prism Software.