Softworrx

After drug treatment, cells were trypsinized, washed once with PBS, centrifuged at 500 rpm for 5 min using a cytospin (Thermo Fisher Scientific, Waltham, MA) and seeded onto poly-l-lysine (PLL)-coated coverslips. The cells were then fixed with 4% para-formaldehyde for 20 min, blocked in 2% BSA/PBS for 30 min, and incubated in primary anti-pH3 and anti-γH2AX antibody overnight (4°C). Cells were then washed three times in 2% BSA/PBS, incubated in Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies for 1 hour at room temperature. DAPI was added to stain nuclei. Cells were imaged on an upright fluorescence microscope (Nikon, Melville, NY), and the data was analyzed using FociCounter (Anna Jucha, University of Wrocław, Poland). Cells were scored based on whether they harbor ≥10 γH2AX foci.
For multipolar and monopolar mitotic spindles staining, cells were washed with PBS and fixed with cold methanol for 20 min at −20°C, followed by incubation with primary antibodiesmin including Cep192 (SPD-2, A. Dammermann, K. Oegema Lab) and α-tubulin (DM1α, Sigma). Images were recorded on a Deltavision microscope at 1 × 1 binning with a 100× NA 1.3 U-planApo objective. Z-stacks (0.2 μm sections) were deconvolved using softWorRx (Applied Precision) and maximum intensity projections were imported into Adobe Photoshop CS4 (Adobe) for analysis.

Images (0.2 μm sections) were acquired at 40×, 60× and 100× magnification of optically bisected germlines^{20 (link)} using a Deltavision, deconvolved, and merged using softWorRx (Applied Precision). Exposure conditions were kept constant for each strain and condition. Germlines regions were determined as described^{50 (link)} and the fluorescence intensity for each region was measured using ImageJ (version 2.0.0) and normalized to the number of nuclei in the given region. 3–8 germlines were quantified for each strain and condition. Student’s t test statistical analyses were performed using GraphPad Prism. Images were further processed using ImageJ and Adobe Photoshop CS6. In Fig. 2, 78–302 mitotic cells, 59–125 transition zone cells and 60–323 pachytene cells were quantified. Quantitative analysis of RAD-51 foci presented in Fig. S5 was performed as described^{51 (link)}.