Anti phospho histone h2a x ser 139 clone jbw301

For immunofluorescence staining, SCC011 cells were grown on cover slips until the reaching of 80% of confluence, fixed with 100% cold methanol on ice, and permeabilized in 0.1% Triton X-100 for 15 min. After permeabilization, cells were blocked with 0.3% BSA/PBS and washed in PBS. Next, cells were incubated with primary γ-H2AX (anti-phospho-Histone H2A.X (Ser 139) clone JBW301, Cat. 05-636, Millipore, Burlington, Massachusetts, USA) and RAD51 (Rad51 (H-92), cod. sc-8349, Santa Cruz Biotechnology) antibodies (for antibodies dilutions see Supplementary Data 3), O/N at 4 °C. Secondary antibody incubation was carried out at room temperature for 1 h, followed by washing in 0.2% Tween/PBS. Images were acquired with a ZEISS LSM 900 Airyscan 2 confocal microscope. For 8-oxo-dG assay, the immunofluorescence staining was performed according to the manufacturer’s instructions, by using the anti-8-oxo-dG antibody (TREVINGEN, cod. 4354-MC-050). The measure of the co-localization of 8-oxo-dG/DAPI signals was performed by counting the number of nuclei in a field. Briefly, after counting the number of nuclei in each field, we determine the number of foci, normalizing for the number of nuclei in each field. The foci in each region were defined by the nuclear staining overlap.

The number of phosphorylated γ-H2AX foci in cell nuclei is an efficient marker for scoring radiation-induced DSBs [36 (link), 39 (link)]. HUVEC were seeded on glass cover slips at 80% of confluence the day before treatment in order to have a monolayer the day of irradiation. The cells were fixed with 2% paraformaldehyde 2 h after irradiation and treated as previously described [36 (link)]. The primary antibody against γ-H2AX (anti-phospho-Histone H2AX (Ser139), clone JBW301, Millipore) was diluted at 1 : 200 in PBS containing BSA/glycine. The secondary antibody tagged to a fluorescent group (Alexa Fluor 594 goat α-mouse IgG, Thermo Fisher Scientific) was applied diluted at 1 : 500 in PBS with BSA/glycine. Cover slips were put on object glasses covered with DAPI/Vectashield and sealed.
Analysis of foci formation was performed using a ZEISS Axioskop40 fluorescence microscope equipped with a ×100 magnification objective. In each sample, the number of foci was counted in 100 cells. The number of foci/cell was determined by the ratio between total number of foci and total enumerated cells.

Antibodies used in this study included KMT5A (cell signalling), CDC20 (Ab190711, and AbCam), PARP1/2 (clone H250, sc-7150, Santa Cruz Biotechnology, Dallas, TX, USA), MDM2 (Clone N-20, sc-813, Santa Cruz Biotechnology), p21 (ab-4, Calbiochem), p53 (pAb-421#OP03, Calbiochem), p53-S15-P (cell signalling), p53-K382-Ac (ab75754, AbCam), H4K20Me1 (Ab9051, AbCam), H4 (07-108, Merck, Darmstadt, Germany), anti-phospho-histone H2AX (Ser139) (clone JBW301, Millipore Corp., Burlington, MA, USA) α-tubulin (clone DM1A, T9026, Sigma, St. Louis, MO, USA), and GAPDH (clone 1E6D9, Proteintech, Rosemont, IL, USA).