U plan s apochromat objective

Stressed and control cells were fixed for at least 30 min in 3.7% formaldehyde. Glass slides were coated with concanavalin A for cell attachment and cells were immersed in Vectashield mounting medium prior imaging. SIM was carried out with a DeltaVision OMX v4 BLAZE (Applied Precision) microscope on an inverted stand, a 4× sCMOS camera (Andor) and a 100×1.4 oil immersion UPlanSApochromat objective (Olympus). API softworx was used for driving the microscope and OMX SI reconstruction was carried out using Centos 4, the OMX SI reconstruction Linux box. A 488 nm DPSS laser and a 561 nm DPSS laser were used to image GFP and mCherry signal, respectively. At least five different Z-stacks (maximum Z-resolution) were recorded for each condition. Cell boundaries if shown (indicated by the white outline in fluorescence microscopy images) were drawn manually.

U2OS LacO cells (a gift from S Janicki) were grown in DMEM supplemented with 8% FBS (Clontech), hygromycin (200 µg/ml), pen/strep (50 µg/ml), and L-glutamine (2 mM). Cells were transfected with the indicated constructs for 48 hr using Fugene HD according to the manufacturer's protocol. Asynchronously growing cells were arrested in prometaphase by the addition of nocodazole (830 nM) for 2–3 hr. Cells plated on 12-mm coverslips were fixed (with 3.7% paraformaldehyde, 0.1% Triton X-100, 100 mM Pipes, pH 6.8, 1 mM MgCl₂, and 5 mM EGTA) for 5–10 min. Coverslips were washed with PBS and blocked with 3% BSA in PBS for 1 hr, incubated with primary antibodies (GFP-booster [Chromotek], rabbit-anti-BUBR1 [Bethyl] and CREST/anti-centromere antibodies [Cortex Biochem, Inc.]) for 16 hr at 4°C, washed with PBS containing 0.1% Triton X-100, and incubated with secondary antibodies (goat-anti-rabbit Alexa Fluor 568 and goat anti-human Alexa Fluor 647) for an additional hour at room temperature. Coverslips were then washed, incubated with DAPI for 2 min, and mounted using antifade (ProLong; Molecular Probes, Eugene, OR). All images were acquired on a deconvolution system (DeltaVision RT; Applied Precision, part of GE Healthcare) with a 100×/1.40 NA U Plan S Apochromat objective (Olympus, Shinjuku, Tokyo, Japan) using softWoRx software (Applied Precision).

Cells were treated with 150 ng/mL Colcemid solution for 30 min in DMEM supplemented with 10% FBS and 50 μg/mL penicillin/streptomycin. Cells were collected by trypsinization and resuspended in hypotonic buffer (20 mM HEPES [pH 8.0], 1 mM MgCl₂, 20 mM KCl, and 0.2 mM CaCl₂) at room temperature for 10 min. Samples were transferred to glass slides using a cytospin, air-dried, fixed in methanol/acetic acid (3:1) at room temperature for 10 min, and then washed twice in PBS. Immunofluorescence using Cenp-C antibody and counterstaining with DAPI were carried out as described above. Imaging was carried out using a Deltavision Core or Elite microscope equipped with a 100×/1.40 NA U Plan S Apochromat objective (Olympus) and a CoolSNAP HQ2 camera (Photometrics) using softWoRx software. Stacks of 20 images at 0.2 μm intervals were taken. All images are projections of deconvolved stacks.