Glass bottom 35 mm u dishes

Amniotic cells, glass-bottom 35 mm u-dishes (Ibidi GmbH, Germany) coated with fibronectin, were co-transfected with H2B-GFP and pmRFP-tubulin expression plasmids (Addgene, MA, USA) using Lipofectamine 3000 transfection reagent and according to the manufacturer's instructions. Live cell imaging was performed 48 hr following transfection under a spinning-disk confocal system Andor Revolution XD (Andor Technology, UK) coupled to an Olympus IX81 inverted microscope (Olympus, UK) equipped with an electron-multiplying CCD iXonEM Camera and a Yokogawa CSU-22 unit based on an Olympus IX81 inverted microscope. Two laser lines at 488 and 561 nm were used for the excitation of GFP and pmRFP and the system was driven by IQ software (Andor Technology, UK). Z-stacks (0.8–1.0 μm) covering the entire volume of the mitotic cells were collected every 1.5 min with a PLANAPO 60×/1.4 NA objective. ImageJ was used to process all the videos.

DLD1 cells, grown on sterilized coverslips inside 35 mm Petri dishes, were transiently transfected with either P-EYFP-N1 or P-EYFP-N1-SPG20 (a kind gift from J Bakowska) using Fugene HD (Roche, Basel, Switzerland) according to the manufacturer's protocol. For SPG20 knockdown, cells grown in glass-bottom 35 mm u-dishes (Ibidi GmbH, Germany) or on sterilized coverslips inside 35 mm Petri dishes, were transfected with Silencer Select siRNA specific to SPG20 (S23057, Ambion, Life Technologies, CA, USA) using Oligofectamine according to the manufacturer's instructions (Invitrogen, Life Technologies, CA, USA). Silencer Select Negative control siRNA (Ambion, Life Technologies, CA, USA) was used as a negative control and was also transfected into cells with Oligofectamine (Invitrogen, Life Technologies, CA, USA). Cells were observed 48–72 hr after transfection.