Dna pk

MSCs were plated on coverslips and allowed to attach before treatment with 1800 ng/mL bleomycin for 4 hours. At the time points indicated in the Results section, cells were fixed with 4% paraformaldehyde. Cells were incubated with antibodies against γH2AX (1:100, Biolegend, London, UK), the catalytic subunit of phosphorylated DNA protein kinase (DNA-PK, 1:8000, Abcam, Cambridge, UK) and Rad51 protein (1:250, Cosmobio Co., Tokyo, Japan) overnight at 4 °C. After several washing steps, secondary antibodies (Alexa Fluor-568 goat anti-rabbit, 1:1000, Invitrogen, Darmstadt, Germany; Alexa Fluor-488 goat anti-mouse, 1:250, Invitrogen; DyLight 650 goat anti-chicken, 1:250, Thermo Fisher Scientific, Karlsruhe, Germany) were then added for 90 minutes at 4 °C. Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). For each condition and time point, 300 cells were automatically detected and assessed by an Axioplan-2 microscope (Zeiss, Jena, Germany) at 40x magnification using Metafer software (Metasystems, Altlussheim, Germany). Each experimental condition was analyzed in triplicate. Foci analysis on a single cell level was carried out using Matlab software (The MathWorks, Natick, Massachusetts, USA).

Antibodies were used according to manufacturer instructions. PARP [#9542, Santa Cruz (Dallas, Texas, USA)], PSA/KLK3 [D6B1, Cell Signalling (Danvers, Massachusetts, USA)], Rad51 (sc-398587, Santa Cruz), DNA-PK [ab70250, Abcam (Cambridge,UK)] and β-Actin (C4: sc-47778) primary antibodies were used in conjunction with HRP conjugated mouse and rabbit secondary antibodies (Life Technologies, USA).