γh2ax antibody

5,000 cells were seeded in 4-chamber coverglass slides (Lab-Tek II) and allowed to adhere overnight. On the following day, cells were irradiated and fixed with 4% formaldehyde for 5 min at indicated time points. Cells were washed twice with 1X PBS, incubated at room temperature in 0.05% triton-X for 15 minutes, washed, and incubated with 1X PBS containing 1% of BSA for 30 min to prevent non-specific binding of the antibody. Finally, cells were incubated in a 1:10 dilution of γH2AX antibody (BD Pharmingen) in 1% BSA for 1 h. Images were taken using an LSM 700 confocal microscope (Zeiss).
Alternatively, for flow cytometry, cells were at harvested at the indicated time points, fixed with 90% ethanol and maintained at −20 °C until the day of experiment. Cells then were centrifuged at 3,000 rpm for 5 min and resuspended in 1% BSA for 30 min. γH2AX antibody (BD Pharmingen) was added to the cells in a dilution of (1:200) and incubated at room temperature for 1 hr. Cells were then analyzed by flow cytometry at an excitation wavelength of 488. Raw data were normalized according to the intensity of control samples (normalized mean intensity = intensity of the sample / the intensity of the corresponding control sample within the same experiment)

For γH2AX analysis of intact BE4max and seBE constructs in the presence or absence of rapamycin, the transfection protocol was performed on HEK293T cells seeded on 6-well plates and transfected at approximately 60% confluency. Parallel analysis of empty vector (pcDNA-EV) or the isolated DNA deaminase domains was carried out. Intact BE4max or seBE constructs and LRcherry2.1-EMX1 sgRNA expression plasmids were transfected in a 2:1 ratio using Lipofectamine 2000 CD (Invitrogen) per well according to manufacturer’s protocol. For seBE experiments, 24 hours after transfection, rapamycin was added to select wells at a final concentration of 200 nM and maintained until the end of the experiment. Cells were harvested 3 days after transfection and stained with γH2AX antibody (BD Pharmigen, 647) for flow cytometry analysis. Cells were gated on FITC and APC using the Fortessa Flow Cytometer (BD Biosciences), and results were analyzed using FlowJo. The gating strategy is exemplified in Extended Data Fig. 8c.

HEK293T cells used for flow cytometry were cultured in Dulbecco’s Modified Eagle Medium (DMEM) media (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO₂ and cells were periodically tested to be mycoplasma negative. The cells were transfected with A3A-INS2 or A3A(E72A)-INS2, or co-transfected with A3A-SPL2_N and A3A-SPL2_C for 24 hours prior to harvest and staining with γH2AX antibody (BD Pharmigen, 647) and flow cytometry analysis. Cells were gated on FITC and APC using the Fortessa Flow Cytometer (BD Biosciences), and results were analyzed using FlowJo. The gating strategy is exemplified in Extended Fig. 8a.
U2OS cells used for immunofluorescent studies were cultured in Dulbecco’s Modified Eagle Medium (DMEM) media (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% CO₂ and cells were periodically tested to be mycoplasma negative. U2OS cells plated on coverslips were transiently transfected with A3A-INS, A3A(E72A)-INS2 or co-transfected with A3A-SPL2_N and A3A-SPL2_C constructs for 24 hours prior to incubation with γH2AX antibody (Millipore Sigma) and immunofluorescent staining with Alexa Fluor 568 (Invitrogen) and DAPI. Stained cells were imaged with a Nikon A1R confocal microscope and analyzed using ImageJ.