Deltavision microscope

Immunofluorescence was conducted as previously described[49 (link)]. For Mad2 and BubR1 staining, cells plated on coverslips were pre-extracted with 0.2% Triton X-100 in PHEM (60 mM PIPES (pH 6.8), 25 mM HEPES, 10 mM EGTA and 2 mM MgCl₂) for 45 s before fixation with 4% paraformaldehyde in PBS. After staining experiments for Aurora A, Aurora B, H3T3ph, H3S10ph, Bub1 and H2AT120ph, cells were fixed directly in 4% paraformaldehyde before extraction. Then, cells were blocked with 1% bovine serum albumin in TBST for 30 min, incubated with primary antibody for 2 h at room temperature, washed with TBST three times and incubated with secondary antibodies for an additional 1 h at room temperature. DNA was stained with 4,6-diamidino-2-phenylindole for 2–3 min. Coverslips were mounted using ProLong antifade (Sigma). Images were acquired on a DeltaVision microscope (GE Healthcare, Buckinghamshire, UK) with a 60× objective lens, NA=1.42, with optical sections acquired 0.2-0.3 μm apart in the z-axis, and were shown as maximal intensity projections. One exposure setting is used within each experiment. Images shown in the same panel have been identically scaled.

HeLa cells were grown on coverslips prior to transfection with either Tax-1 or empty expression plasmid or SNX27 or scramble siRNA. 24 hours post-transfection the cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature followed by permeabilization with 0.1% Triton X-100 for 10 minutes at room temperature. Cells were then blocked with 5% bovine serum albumin (BSA) in 1× PBS for 1 hour at room temperature. Cells were then treated with GLUT1 (Abcam, AB15309; 1:100) and LAMP1 (Abcam, AB25630; 1:300) primary antibodies diluted in a 2% BSA 1× PBS solution overnight at room temperature. Alexa Fluor 568 goat anti rabbit and Alexa Fluor 488 goat anti mouse secondary antibodies (Life Technologies; 1:500) were diluted in 2% BSA 1× PBS solution and incubated on the cells for 1 hour at room temperature. Coverslips were mounted onto slides using VECTASHIELD mounting medium with DAPI (Vector Laboratories) per manufacturer’s instructions. Deconvolution images were acquired at room temperature using an inverted DeltaVision microscope (GE) with an oil immersion 60× objective lens. Subsequent colocalization analysis to determine Pearson’s coefficient of correlation and processing of images were performed using the DeltaVision software (GE).

HCT116 cells were cultured in a glass-bottomed dish (MatTek) containing medium without phenol red. To visualise nuclei in live cells, 0.5 μM SiR-DNA (Spirochrome) was added to the medium before observation. Live-cell imaging was performed using a DeltaVision microscope equipped with an incubation chamber and a CO₂ supply (GE). Image analysis and quantification was performed using the Volocity software (PerkinElmer).

Live cell imaging of meiotic events was performed as described in [17 (link),19 (link)]. Briefly, 1 ml mating culture was harvested at 5000 rpm for 30 s. Pellets were resuspended in 250 µl ME media. 10 µl of cell suspension were spread on top of 2% agarose pad made with liquid sporulation media and sealed with VaLaP (Vaseline/ Lanolin/ Paraffin in a ratio 1 : 1 : 1 by weight). Live cell imaging was performed at 25°C (pre-calibrated chamber) on a Delta Vision Microscope (Applied Precision, GE Healthcare, Issaquah, WA) equipped with Olympus 60x/1.40 Plan-Apo objective lens, solid-state illuminator, and 12-bit Photo metrics CoolSNAP_HQ2 Charged-coupled device (CCD) camera. Different filter sets and exposure timings were used to excite and detect fluorescent proteins (supplementary table S1). 13 optical z-sections of 0.5 µm step size were acquired for each field at 10-minute intervals over 8 h. Images were acquired, deconvolved and all z-stacks projected into a single-plane as maximum intensity projection by SoftWoRx Version 5.5.1 (GE, Issaquah, WA) software. Finally, the projected fluorescence images were fused with transmitted light images. Downstream processing and image analysis were done using Fiji (ImageJ), an open source image analysis software [98 (link)]. Detailed description of image analysis is given in [17 (link),19 (link)].