Sp8uv microscope

Confocal imaging of smiFISH and IF-smiFISH samples was performed on an SP8UV microscope (Leica) equipped with a 633-nm HeNe laser, a 561-nm DPSS laser, a 488-nm argon laser and a 405-nm laser diode. A ×63 oil immersion objective (NA 1.4) was used and images were taken by using the hybrid detector photon-counting mode. The laser power for all acquisitions and laser lines was set to 10%. All images acquired have a bit depth of 8 bit and a pixel resolution of 70 nm. The z-stacks were taken with a z-spacing of 300 nm for a total of 4–6 µm. Image processing was performed using the Fiji/Image J software. All images were processed the same way. In detail, the channels of the different images were split and grey values were adjusted to better visualise the spots in the cytoplasm. The nuclear signal in the green channel (TAF10 or TAF8 IF) was removed by masking the nucleus and using the “clear” option. Finally, the processed channels were merged again. For IF-smiFISH, one cell of an image was cropped and one representing z-slice per cell was chosen. For smiFISH, maximum intensity Z-projections of individual images were made and one cell per resulting image was cropped as the representative image. In addition, one single IF or smiFISH spot from the corresponding cells was cropped as well.

Immunofluorescence was performed as described [69 (link)]. Briefly, cells were either fixed in methanol for 5min at −20°C or in 4% PFA (in PBS with 0.2% Triton X-100), immunostained with indicated primary antibodies Secondary antibodies conjugated to Alexa fluorochromes (Thermo Fisher Scientific-Invitrogen) were used. F-actin and DNA were, respectively, stained with phalloidin-Atto647 and DAPI (Sigma-Aldrich). For analyses of mitotic parental cells, cells were seeded on L shape CYTOOchips™, as recommended by the manufacturer. Confocal microscopy was performed using a Leica SP8-UV microscope equipped with Leica 63x HCX PL APO 1.4 oil CS2 objective. For quantitative comparison, fluorescent staining of respectively KIF11 or AURKA were acquired using same laser power and conditions. Stack images were acquired and maximum intensity projection (MIP) of images (keeping same number of grey levels between images) were analyzed with Image J software.