Csu x1 spinning disk

RPE1 CRISPRi or CRISPRa cells were infected with lentivirus as described above. Subsequently, cells were selected using 10 μg/ml puromycin for 7 days. The cells were seeded in 96-wells glass bottom dishes (Matriplate, Brooks). Immediately prior to imaging the medium was replaced by Leibovitz’s L-15 (GIBCO) CO₂-independent medium supplemented with the indicated concentrations of rigosertib. The cells were imaged using a Yokogawa CSU-X1 spinning disk confocal attached to an inverted Nikon TI microscope with Nikon Perfect Focus system, CFI Plan Apochromat 20X NA 0.75 objective, an Andor iXon Ultra 897 EM-CCD camera, and Micro-Manager software (Edelstein et al., 2014 (link)). Cells were imaged every 15 min for 10 hr. For RPE1 CRISPRa cells, nuclear envelope breakdown and reformation was determined by nuclear localization of the scFv-GFP-NLS and was defined as mitotic entry and exit, respectively. For RPE1 CRISPRi cells, which do not express scFv-GFP-NLS, the duration of mitosis was determined as the moment of mitotic cell rounding until the moment of cleavage furrow ingression, as determined by fluorescence of BFP expressed from the sgRNA expression construct.

Flies were mounted in oil (refractive index 1.334, Zeiss Immersol) between glass coverslips spaced with double-sided tape as shown in Fig. 6C and described previously (61 (link)). Mitochondrial transport recordings were performed on an inverted spinning disk microscope (Nikon Ti-Andor Revolution-Yogokawa CSU-X1 Spinning Disk) fitted with a Nikon 60× objective (Plan Apo, NA 1.27, W) and incubation chamber (Okolab, 25 °C). TdTomato was excited with 561 nm laser light and a dual-band bandpass filter was used for emission (FF01-512/630-25, Laser 2000). The 10-min long transport recordings consisted of 10-µm thick stacks, to account for wing movement, taken every 2 s. Mitochondrial time lapse recordings were registered using the StackReg plugin (ImageJ) and in-house Igor pro code was used to generate kymographs and analyze mitochondrial transport time lapse recordings as described previously (35 (link), 62 (link), 63 (link)).