Anti γh2axser139 antibody

In order to confirm the induction of mitotic arrest and/or DNA damage after Plk1 inhibition, immunofluorescence experiments using the mouse monoclonal anti‐pHH3 (Ser10) antibody (1 : 2000, Merck Millipore, no. 05‐806) and anti‐γ‐H2AX (Ser139) antibody (1 : 500, Merck Millipore, no. 05‐636‐AF488) were performed. Seventy‐two hours (γ‐H2AX) or 24 h (pHH3) after treatment with volasertib (0–50 nm), cells were fixed with ice‐cold methanol, permeabilized with 0.1% Triton X‐100/PBS, and blocked with 1% BSA/PBS for 1 h. Next, cells were incubated overnight with the primary antibody at 4 °C, followed by 1‐h incubation at room temperature with the secondary antibody, that is, donkey anti‐mouse IgG Alexa Fluor^® 555 conjugate (1 : 1000, Thermo Scientific). Slides were counterstained with DAPI and mounted. Images of sections stained with the anti‐pHH3 antibody were taken using an Olympus BX51 standard research fluorescence microscope (Olympus, Aartselaar, Belgium), equipped with an Olympus DP71 digital camera (Olympus). Sections stained with the anti‐γ‐H2AX antibody were visualized with an Evos Cell Imaging System (Thermo Scientific). The percentage of positive pHH3 cells and the amount of γ‐H2AX foci per cell were counted using imagej software.

The γH2AX and pATM immunofluorescence protocol for assessing DSB recognition and repair was described elsewhere [26 (link)–28 (link)]. Briefly, cells were fixed in paraformaldehyde for 15 min at room temperature and permeabilised in detergent solution for 3 min. Primary antibody incubations were performed for 1 h at 37 °C. Anti-γH2AX^ser139 antibody (#05-636 Merck, Molsheim, France) was used at 1:800, the monoclonal anti-mouse anti-pATM^ser1981 (#05-740 Merck) was used at 1:100. Incubations with anti-mouse fluorescein secondary antibodies provided by Sigma-Aldrich (L’Isle d’Abeau Chesnes, France) were performed at 1:100 at 37 °C for 20 min. Slides were mounted in 4′,6′ Diamidino-2-phenyl-indole-stained Vectashield (Vector Laboratories, Burlingame, CA, USA) and cells were counted using an X100 objective with a fluorescence BX51 Olympus microscope (Olympus-France, Rungis, France). For each of the three independent experiments, 100 nuclei were analysed. The patented procedures of foci scoring have been detailed elsewhere [28 (link)].

The immunofluorescence protocol and nuclear protein foci scoring was described elsewhere [43 (link),54 (link)]. Anti-γH2AX^ser139 antibody (#05-636; Merck Millipore, Burlington, VT, USA) was used at 1:800. The monoclonal anti-mouse anti-pATM^ser1981 (#05-740) from Merck Millipore was used at 1:100. Incubations with anti-mouse fluorescein (FITC) and rhodamine (TRITC) secondary antibodies were performed at 1:100 at 37 °C for 20 min. By following the same procedure, micronuclei were scored on the same slides by using 4′,6′Diamidino-2-Phényl-indole (DAPI)-counter staining. Foci and micronuclei were scored by eye with an Olympus BX51 fluorescence microscope. For each of the three independent experiments, 100 nuclei were analyzed. The patented procedures of foci scoring have been detailed elsewhere [26 (link)]. It is noteworthy that post-irradiation times indicated in the text represent an equal period of time of the incubation of cells at 37 °C without any genotoxic stress (i.e., under standard culture conditions). Such a period is currently considered to be a time for repair [43 (link),54 (link)].

The immunofluorescence protocol and nuclear protein foci scoring are described elsewhere [29 (link),30 (link)]. Four antibodies against USH1 proteins were used at 1:100: the polyclonal anti-rabbit anti-cadherin like 23 (#ab131135), the monoclonal anti-rabbit anti-myosin VII A (#ab150386), the polyclonal anti-rabbit anti-USH1G (#ab150820), and the monoclonal anti-USH1C (#ab56812), all from Abcam (Cambridge, UK). Anti-γH2AX^ser139 antibody (#05-636; Merck Millipore, Burlington, VT, USA) was used at 1:800. The monoclonal anti-mouse anti-MRE11 (#56211) from QED Bioscience (San Diego, CA, USA) and the monoclonal anti-mouse anti-pATM^ser1981 (#05-740) from Merck Millipore were used at 1:100. Incubations with anti-mouse fluorescein (FITC) and rhodamine (TRITC) secondary antibodies were performed at 1:100 at 37 °C for 20 min. Slides were mounted in 4′,6′diamidino-2-phenyl-indole (DAPI)-stained VECTASHIELD (CliniSciences, Nanterre, France) and examined with an Olympus BX51 fluorescence microscope.