γh2ax

Histology and immunohistochemical analysis were done as described by us in [12 (link)]. Briefly, tissues were overnight fixed at 4°C, in 4% PFA, embedded in paraffin and 5 μm sections were prepared. Slides were deparaffinised and incubated with primary antibodies including βcatenin (610154, BD Transduction Labs, CA, USA); PCNA, Plzf (sc-56: PCNA; sc-22839: Plzf; Santa Cruz Biotechnology, CA, USA); Foxo1, LEF1, TCF1 (#2880: Foxo1; #2230: LEF1; #2203: TCF1; Cell Signaling Technology, MA, USA); Cyclin D1, SCYP3, Stra8 (ab16663: Cyclin D1; ab15093: SCYP3; ab49602: Stra8; Abcam, Vic, Australia); GCNA [49 (link)]; γH2AX (#05-636: γH2AX; Millipore, MA, USA), αSMA (c6198, Sigma, MO, USA) and AlexaFluor secondary antibodies (1:250; Jackson ImmunoResearch Labs, PA, USA). For detection of apoptotic cells, TUNEL assay was performed on paraffin sections as per the instructions provided with the kit (Millipore). For cell counting images at 20x magnification were taken with Olympus DP72 microscope keeping same exposure and gain for both control and mutant tissues. Each testis was divided into four sections and at least two images were randomly selected from each section from minimum three control and mutant animals. The cells were counted using ImageJ (National Institute of Health, USA).

The antibodies used in this study are BRCA1 (44 (link)) (provided by Raimundo Freire), CTIP (A300-488A, Bethyl Laboratories), BLM (A300-110A, Bethyl Laboratories), 53BP1 (NB100-304, Novus Biologicals), β-Actin (MA1-140, Thermo Fisher Scientific), TOPBP1 (44 (link)) (provided by Raimundo Freire), RRM2 (17 (link)) (provided by Raimundo Freire), RPA2-pS4/8 (A300-245A, Bethyl Laboratories), DNA-PKcs-pS2056 (PA5-78130, Thermo Fisher Scientific), DNA-PKcs (A300-516A-T, Bethyl Laboratories), Vinculin (#4650, Cell Signaling), RAD51 (PC130, Calbiochem), RAD52 (5E11E7, Thermo Fisher Scientific), γH2AX (JBW301, Sigma Millipore), γH2AX (A300-081A, Bethyl Laboratories) and E2F1 (sc-251, Santa Cruz Biotechnology).

Fibroblasts were grown on coverslips for 24 h, fixed with 4% PFA, washed, and blocked for 1 h in 10% goat serum/PBS/0.1% Triton. Independent coverslips were incubated with p21 (1:200; Calbiochem OP64), cleaved caspase 3 (1:1000; Cell Signaling 9661), γH2AX (1:1000; Millipore 05-636), or RPA2 (1:200; Calbiochem NA19L) antibodies. After overnight incubation in primary antibodies, signals were detected using fluorescently conjugated antibodies and costained with DAPI. For all antibodies except cleaved caspase 3 and p21, cells were pre-extracted for 5 min on ice with ice-cold buffer (25 mM HEPES at pH 7.4, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl₂, 300 mM sucrose, 0.5% Triton X-100) before fixation to remove soluble proteins and detection of chromatin-bound fractions. For γ-H2AX staining, cells were pulsed with 20 µM EdU (Sigma 900584) for 1 h before fixation. EdU was detected subsequent to immunofluorescence using the click reaction and azide-Alexa fluor 488 (Thermo Fisher A10266).

Organoids were washed once in ice-cold-PBS, were collected in 1 mL ice-cold cell recovery solution (Corning, #354253) and 1 mL of ice-cold PBS in a 15 mL tube, and were incubated on ice for 10 minutes. Organoids were washed once with ice-cold PBS and were fixed by 4% PFA (#1004965000, Merck Millipore) for 20 min at RT and stored in PBS at 4 °C. For staining, organoids were transferred into 1.5 mL Eppendorf tubes. Organoids were permeabilized with PBS buffer containing 10% DMSO, 2% Triton X-100, and 10 g l⁻¹ BSA for 4 h at 4 °C. Organoids were stained with primary antibodies (γH2AX 1:400, Sigma, #05-636, and Ki67 1:200, Abcam, #ab15580) overnight, Alexa fluorophore-conjugated secondary antibodies (Invitrogen) for 4 h and with DAPI for 1 h at 4 °C. Imaging was performed using a SP8 confocal microscope (Leica Microsystems). Light microscopy was performed using EVOS M5000 imaging system (Invitrogen).

Samples grown over Nunc Lab-Tek chambers (Thermo Fisher Scientific, Waltham, MA, USA) were washed with PBS, fixed with 4% paraformaldehyde for 15 min, washed three times with PBS and blocked with PBS 1% BSA-0.3% Triton for 60 min. Samples were then incubated overnight at 4 °C with primary antibody against BAX-6A7 (BD Bioscience, Franklin Lakes, NJ, USA), activated Caspase 3 (Cell Signaling, Danvers, MA, USA) or γH₂AX (Cell Signaling, Danvers, MA, USA) in PBS 1% BSA-0.3% Triton.
After three washes with PBS, samples were incubated with the corresponding FITC-conjugated (Activated Caspase-3 or γH₂AX) or TRITC-conjugated (BAX-6A7) secondary antibody (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 h. Samples were washed three times with PBS and cell nuclei were finally stained with DAPI (2 μg/mL). Images were captured using a ZEISS Axio Observer (ZEISS, Oberkochen, Germany) microscope and analyzed using the Carl Zeiss Microscopy GmbH’s ZEN 3.0 software.

An antibody against GAPDH (MAB374) was from Millipore; Calnexin (ab22595) was from Abcam; Tubulin (DM1A) was from Sigma; CHMP2A (10477–1-AP) was from Proteintech; IST1 (51002–1-AP) was from Proteintech; CHMP7 (16424–1-AP) was from Proteintech; GFP (7.1/13.1) was from Roche; mCherry (ab167453) was from Abcam; HA.11 (16B12) was from (Biolegend); γH2AX (05–636) was from Sigma; 53BP1 (NB100-305) was from (Novus Biologicals). Anti-CDK1 substrate antibodies were from Cell Signaling Technology (9477S and 2325S). Anti-Histone H3 pS10 was from Cell Signaling Technology (9701S). Anti-LEM2 was from Sigma (HPA017340); anti-HSP90 (F8) was from Santa Cruz; anti-Emerin (10351–1-AP) was from Proteintech. Anti-LAP1 (21459–1-AP) was from Proteintech. Alexa conjugated secondary antibodies were from Invitrogen and HRP-conjugated secondary antibodies were from Millipore. IRDye 800 CW (925–32210) and IRDye 680 RD (925–68071) were from LI-COR Biosciences. Anti-peptide antibodies against CHMP7 pSer3 (3891 and 3892) were generated by immunisation of rabbits with KLH-conjugated peptides (MWpSPEREAEAPAGGC) by GenScript Biotech (Netherlands) B.V.

Snap-frozen tissue sections derived from seven organotypic breast tumors or MCF-7 cells were fixed in 4% paraformaldehyde (PFA) (Sigma-Aldrich), and incubated with a primary antibody to phospho-histone H2AX (pSer¹³⁹) (γH2AX, 1:700, Sigma-Aldrich) for 16 h at 22°C. Anti-rabbit 647 or 548 Alexa Fluor-conjugated was used as a secondary antibody (Molecular Probes, Invitrogen), and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Abbott Laboratories, Abbott Park, IL, USA). Slides were scored by fluorescence microscopy using an AxioImager Z1 microscope (Carl Zeiss, Göttingen, Germany) and photographed images were arranged with Photoshop. For each sample, 100 cells were analyzed and the H2AX phosphorylation index was calculated as a percentage of γH2AX-positive cells
[26 (link)].