Mitotic index and pH2AX foci quantification

Mitotic index and pH2AX foci were quantified by platting 1 × 10⁵ cells onto Falcon^® 8-chamber culture slides (cat. #354118). Cells were allowed to attach overnight and were fixed in 4% paraformaldehyde solution (Electron Microscopy Sciences, cat. #100503-917). Standard immunofluorescence was carried out for α-tubulin (Cell Signaling, cat. #8058) and pH2AX (Cell Signaling, cat. #9718) using secondary anti-rabbit antibody Alexa Fluor 594 conjugate (Cell Signaling cat. #8889), and DAPI staining (Sigma, cat. #D9542) for DNA counterstaining. Slides were photographed using a Zeiss AxioObserver.Z1 fluorescence microscope and at least 5 different fields were captured for each biological replicate. A total of 4 independent biological replicates were photographed per each cell type analyzed. Using image J software, version 1.52a, a total number of cells per field was estimated based on the number of DAPI-positive nuclei. A total number of mitotic cells was estimated based on recognition of mitotic figure visualized by DAPI and α-tubulin staining. A total number of cells positive for pH2AX was estimated, when the number of foci was larger than 20 per nuclei. The percentage of cells in mitosis, cells positive for pH2AX and mitotic cells positive for pH2AX (Tyk-nu cells only) was estimated based on a total number of cells. Data were represented as average ± SE.