Simplepci software

The FPP method refers to an assay previously described by Lorenz et al.^{25 (link),26 (link)}. HEK293 cells transiently transfected with plasmids were cultured to 60–70% confluence on coverslips onto glass coverslips which were put on the bottom of multi-well dishes. After removal of culture medium, cells were washed three times for 1 min each in KHM buffer (110 mM potassium acetate, 20 mM HEPES, 2 mM MgCl₂) at room temperature. Cells were kept in KHM buffer and directly imaged microscopically in a “pre-permeabilization” state. To permeabilize the plasma membrane, KHM buffer containing 20 μM digitonin was added and images were recorded as the “post-permeabilization” state. At this point, cells were washed with KHM buffer three times and treated with 50 μg/mL proteinase K in KHM buffer. Coverslips were removed and cells imaged using confocal laser scanning microscopy. For each condition, multiple coverslips were imaged (≥30 cells per coverslip) under identical settings (NIS-Elements Viewer 4.20; Nikon). Fluorescence intensity was determined using SimplePCI™ software (Hamamatsu).

GFP-tagged CSB expression was induced by supplementing the growth media with 5–10 ng/ml doxycycline. Mitotic duration was performed in 12-well plates. An hour prior to imaging, complete DMEM media was changed to CO₂-independent media without phenol red supplemented with 5–10 ng/ml doxycycline. Phase contrast images of cells were then acquired at 37°C using a Zeiss Axio observer Z1 microscope controlled by SimplePCI software (Hamamatsu), equipped with Ocra 03GO1 CCD camera (Hamamatsu) and a Plan-Apochromat 10×/0.45 DIC H objective. The live imaging was performed over 48 h with multiple recording positions per well with 5 min interval between image acquisitions. This represents ±600 image acquisitions per position over the 48 h imaging period. Images were processed using ImageJ software and analysed using mitotic duration plugin. The image frames between mitotic cell rounding (start of mitosis) and appearance of the cleavage furrow (transition from mitosis to cytokinesis) were counted and the number of frames were multiplied by 5 min, hereby resulting in the mitotic duration in minutes for that particular cell. Approximately 50 events were counted for each cell line used.

Intact embryos were fixed in 2.5%PFA/ethanol [95 (link)] or methanol/acetone [96 (link)] for all immunofluorescence except for those probed with monoclonal antibody H5, which was fixed in methanol/formaldehyde [95 (link)]. Primary antibodies used were: anti-H3K4me2 (CMA30) 1:1000 END Millipore], anti-P-granules [OIC1D4, 1:5 [96 (link), 97 (link)])], anti-H3K36me3 [(CMA333), 1:1000 [95 (link)]], anti-Ser2p RNA pol II CTD (H5, 1:500, Covance MMS-129R), anti-GFP (1:1000, Novus NB600-308), anti-AMA-1 (1:10,000, Novus 38520002), and anti-FLAG (M2, 1:1000, Sigma F1804). Secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-mouse (1:500, Invitrogen R37114) and Alexa Fluor 594-conjugated goat anti-rabbit (1:500, Invitrogen R37117). Samples were mounted in ProLong Gold anti-fade reagent (Life technologies, P36934) and observed under a fluorescence microscope (Leica DMRXA; Hamamatsu Photonics, Hamamatsu, Japan) with Simple PCI software (Hamamatsu Photonics). Image J was used for quantification of raw immunofluorescence intensity [98 ].