Softworx 5

Cells were transfected with EB3-GFP and RFP-CenpB to label assembling MTs and centromeres, respectively (25 (link)). Movies of MT assembly were collected over 30 sec at 500 ms intervals 10 min after drug application (0.5 µM ST-11, 3 µM ST-11, or 1 µM nocodazole) on a Deltavision microscope system (Applied Precision, Issaquah, WA). Images were deconvolved using SoftWorx 5.0 (Applied Precision). MT assembly rates were scored in interphase cells using Fiji TrackMate (26 (link)).

For MT visualization, cells were pre-extracted in warm PEM buffer (100-mM PIPES, pH 6.9, 2.5-mM EGTA, 5-mM MgCl₂) supplemented with 0.5% Triton X-100 for 1 min and fixed with 1% glutaraldehyde in PEM for 10 min. Cells were then stained a monoclonal antibody against α-Tubulin (T9026; Sigma-Aldrich) followed by a secondary antibody conjugated with Alexa Fluor 594 or 647 (Thermo Fisher Scientific).
For PRC1 visualization, cells were pre-extracted in warm PEM buffer (100-mM PIPES pH 7, 1-mM EDTA, 1-mM MgCl₂) supplemented with 0.5%Triton X-100 for 30 seconds and fixed with 3.2% paraformaldehyde and 0.1% glutaraldehyde in warm PEM buffer. Cells were then stained with a rabbit polyclonal antibody^{29 (link)} at 1:1000 followed by a secondary antibody conjugated with Alexa Fluor 594 (Thermo Fisher Scientific). Staining for different antigens was done sequentially. Chromosomes were stained with Hoechst 33343 at 1 μg/ml.
Images of fixed cells were collected on the same microscope as live-cell recordings at 73 or 110-nm XY pixels and 200-nm Z-steps. All images were deconvolved with the SoftWoRx 5.0 (Applied Precision) and objective lens-specific point spread function. Precise Axial and Equatorial views of the spindle were generated by rotating the volume in 3-D to orient the spindle axis defined by the 3-D coordinates of both spindle poles.