Adherent HeLa cells were grown in logarithmic phase in F12:DMEM 50:50 medium (Invitrogen, Carlsbad, CA) with 5% CO2 at 37°C. To obtain synchronized HeLa cells in mitosis, cycling cells were treated with 300 nM nocodazole (Sigma-Aldrich, St. Louis, MO) for 18 h. For in vivo Cdk inhibition, cells were incubated with 2.5 μM roscovitine (Rosco; Sigma-Aldrich) for 1 h before cell harvesting. For in vivo phosphatase inhibition, cells were incubated with 175 nM OA (Sigma-Aldrich) for 30 min before cell harvesting. For phosphatase gene knockdowns, cells were transfected with Dharmacon (Lafayette, CO) ON-TARGETplus SMARTpool small interfering RNA (siRNA; NonTargeting cat. no. D-001810-10, PPP1CA cat. no. L-008927-00, PPP2CA cat. no. L-003598-00, PPP2R1A cat. no. L-010259-00, PPP2R2A cat. no. L-004824-00, PPP2R2B cat. no. L-003022-00, PPP2R2C cat. no. L-019167-00, PPP2R2D cat. no. L-0322298-00, PPP2R3A cat. no. L-017376–00, PPP2R3B cat. no. L-019459-00, PPP2R4 cat. no. L-005214-00, PPP2R5A cat. no. L-009352-00, PPP2R5B cat. no. L-009366-00, PPP2R5C cat. no. L-009433-00, PPP2R5D cat. no. L-009799-00, PPP2R5E cat. no. L-008531-00, PPP3CA cat. no. L-008300-00, PPP4C cat. no. L-008486-00, PPP5C cat. no. L-009259-00, PPP6C cat. no. L-009935-00) at 50 nM using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) for 48 h before harvesting or fixation.
Mitotic Microtubule Copelleting Assay
Mitotic microtubule copelleting assays were performed as previously described (Mack and Compton, 2001
). Mitotic HeLa cells were harvested by mitotic shake-off, washed in PBS plus 20 μg/ml cytochalasin B (Sigma-Aldrich) twice, washed with KHMD (78 mM KCl, 50 mM HEPES, pH 7.0, 4 mM MgCl2
, 2 mM EGTA, 1 mM DTT, 20 μg/ml cytochalasin B) plus Halt phosphatase inhibitor (Thermo Scientific, Waltham, MA) once and resuspended in KHMD plus protease inhibitors leupeptin/pepstatin/chymostatin, 1 μg/ml, plus phosphatase inhibitors (as indicated). Cells were Dounce-homogenized, and the extract was cleared by ultracentrifugation at 38,000 rpm for 15 min. All steps were carried out at 4°C unless otherwise noted. Cleared lysates were supplemented with 5 μg/ml latrunculin B (Sigma-Aldrich) and 2.5 mM ATP. Microtubule polymerization reactions were carried out in the presence of control vehicle DMSO or 10 μM taxol (Sigma-Aldrich) at 33°C for 30 min. Polymerization reactions were layered onto a 50% wt/vol sucrose/KHMD cushion supplemented with 10 μM taxol for reactions with taxol-stabilized microtubules. Layered reactions were centrifuged for 2 h at 39,000 rpm in a TLS-55 (Beckman Instruments, Brea, CA) swinging bucket rotor. Samples from the supernatant were placed in an equal volume of 2× Laemmli sample buffer. The microtubule copelleting fractions were washed twice with KHMD buffer and resuspended in 1× Laemmli sample buffer. Supernatant (S) and pellet (P) samples were boiled for 5 min at 90°C, run on an 8% Tris-glycine gel, transferred onto Immobilon-P membrane (Millipore, Billerica, MA), and probed with indicated antibodies.
Mitotic Extract Treatments
For in vitro Cdk inhibition, extracts were incubated in 10 μM Rosco. For in vitro PPP2 phosphatase inhibition, extracts were incubated with 10 nM OA. For in vitro APC/C phosphorylation, cyclin B/Cdk1 (Promega, Madison, WI) was used as described by the manufacturer. For in vitro APC/C dephosphorylation, lambda phosphatase (NEB, Ipswich, MA) was used according to the manufacturer's instructions.
HeLa cells were transfected with control or indicated siRNA (ON-TARGETplus SMARTpool siRNA, Dharmacon) for 48 h, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100/PBS, and stained with 0.5 μg/ml Hoechst 33342, rat anti-α-tubulin (Serotec, Oxford, United Kingdom) and either rabbit anti-Cdc27, Eg5, or cyclin B or mouse anti-centrin. For acute OA treatment, cells were treated with 175 nM OA for 13 min before fixation and staining. Slides were mounted with ProLong Gold anti-fade reagent (Invitrogen), and projection images (10-μm stacks captured every 0.5 μm) were captured with a Zeiss Axio Imager.Z1 microscope (Thornwood, NY) equipped with a CoolSNAP HQ camera (Photometrics, Tucson, AZ) and operated with SlideBook 4.2 (Intelligent Imaging, Denver, CO) at 63× (NA 1.4) at room temperature. One hundred cells were analyzed to determine the percentage of cells with Cdc27 spindle pole localization in control, indicated siRNA, and acute OA-treated cells. A 2 × 2-μm square was drawn around each of 20 spindle poles from control, indicated siRNA, or acute OA-treated cells, and the mean fluorescence intensity of Cdc27 or cyclin B spindle pole staining was plotted as arbitrary units (AU). Additionally, intensity measurements were taken along an axis intersecting the two spindle poles and the fluorescence intensity was graphed as arbitrary units (AU). Image processing was performed using Adobe Photoshop CS2 (version 9.0.2; San Jose, CA).
The following antibodies were used: mouse anti-Cdc27 (Western blot), Eg5 (BD Transduction Laboratories, Lexington, KY), mouse anti-Cdh1 (Neomarkers, Fremont, CA), rabbit anti-Cdc20, cyclin D, cyclin B (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-PPP1CA, PPP2CA, PPP2R1A, and PPP3CA (Upstate), rabbit anti-PPP2R2B (Bethyl Labs, Montgomery, TX), rabbit anti-PPP4C (Novus Biologicals, Littleton, CO), mouse anti-PPP5C (BD Transduction Laboratories), rabbit anti-PPP6C (Chemicon, Temecula, CA), rat anti-α-tubulin (Serotec), goat anti-GAPDH (Novus Biologicals), rabbit anti-pericentrin (Abnova), and rabbit anti-Emi1 (Zymed Laboratories, South San Francisco, CA). Rabbit anti-Apc8 was a gift from H. Yu (University of Texas Southwestern Medical Center, Dallas, TX). Rabbit anti-Cdc27 (immunofluorescence) was a gift from J. M. Peters (Research Institute of Molecular Pathology, Vienna, Austria). Mouse anti-centrin was a gift from J. L. Salisbury (Case Western Reserve University, Cleveland, OH). FITC and Cy3-conjugated secondary antibodies (AffiniPure) were from Jackson ImmunoResearch (West Grove, PA).