Depletion of nucleophosmin leads to distortion of nucleolar and nuclear structures in HeLa cells.

EXPERIMENTAL Cell culture, siRNA (small interfering RNA) transfection and rescue assay HeLa cells were cultured in DMEM (Dulbecco's modified Eagle's medium; Gibco BRL) supplemented with 5% (v/v) FBS (fetal bovine serum) at 37 °C in a humidified incubator with 5% CO 2 . Cells were treated with 2.5 mM thymidine (Sigma) for 16 h, washed and released into fresh medium for 8 h, and then treated with thymidine for a further 16 h to obtain cells uniformly blocked at the G 1 –S-phase boundary. At 12 h after release from the second thymidine block, cells were harvested for further analysis. A double-stranded siRNA sequence (5′-AGAUGAUGAUGAUGAUGAUTT-3′) was used to knockdown human NPM. An siRNA sequence specific for GL2 luciferase gene was used for control RNAi (mock) [ 13 ]. The siRNA sequences for fibrillarin and nucleolin have been published previously [ 13 , 15 ]. siRNA transfection was performed according to manufacturer's protocol (Invitrogen). For the siRNA rescue assay, we constructed an NPMr [RNAi-refractory GFP (green fluorescent protein)-NPM] vector by introducing three silent mutations into the vector through changing the nucleotide sequence (residues 671–682) of NPM to GA C GATGA C GA C (the underlined nucleotides indicate silent mutations). Site-directed mutagenesis was performed by PCR and confirmed by sequencing. The RNAi-refractory construct was transfected into 24-h-old HeLa cell cultures using FuGENE™ 6 (Roche) before ∼6 h of siRNA transfection. At 24- and 48-h post-transfection, cells were harvested and used for further experiments.

Microtubule-polymerization assay

HeLa cells were transfected on to coverslips with mock or NPM siRNAs. At 48 h after transfection, cells were transferred to ice-cold medium supplemented with 10 mM Hepes (pH 7.25) for 30 min for the depolymerization of microtubules. Cells were then transferred to medium at 37 °C for 0, 30 and 60 s and 5 min so that microtubules could be allowed to re-grow and these were then fixed immediately in 99.8% methanol at −20 °C.

Western-blot analysis

Western-blot analysis was performed using standard methods. The mouse monoclonals against B23/NPM (Santa Cruz Biotechnology), α-tubulin (Calbiochem) and β-actin (Sigma) were used at 1:500 to detect NPM, tubulin and β-actin respectively. Goat anti-(lamin A/C) (Santa Cruz Biotechnology) was used at 1:100 to detect lamin A/C, and mouse anti-nucleolin (Abcam) and rabbit anti-fibrillarin (Abcam) at 1:250 were used to detect nucleolin and fibrillarin respectively. Secondary antibodies conjugated to alkaline phosphatase (anti-mouse from Leinco Technologies; anti-rabbit and anti-goat from Vector Laboratories) were used for immunoreactions, which were finally detected by NBT (Nitro Blue Tetrazolium)/BCIP (5-bromo-4-chloroindol-3-yl phosphate) solution (Roche) in alkaline phosphatase buffer [100 mM Tris/HCl (pH 9.5), 100 mM NaCl and 1 mM MgCl 2 ].

Indirect immunofluorescence microscopy

HeLa cells grown on coverslips and fixed with 4% (w/v) paraformaldehyde at 37 °C or 99.8% methanol at −20 °C were incubated with primary antibodies. The antibodies used in the present study were as follows: goat anti-NPM and anti-(lamin A/C) at 1:50; mouse monoclonal antibodies against α-tubulin, β-actin, Ki-67 (Dako) and nucleolin at 1:100; mouse monoclonal antibody against NPM (Santa Cruz Biotechnology) at 1:1000; human anti-centromere autoantibody (CREST; Cortex Biochem) at 1:1000; and a rabbit polyclonal anti-fibrillarin antibody (Abcam) at 1:100. Immunofluorescence staining was performed using standard methods. DNA was stained with Hoechst 33342 (Sigma). All images were acquired as z -stacks with 0.2-μm spacing using a ×100, 1.3 NA oil objective on an IX-70 microscope (Olympus) and processed by iterative constrained deconvolution (SoftWorx, Applied Precision Instruments). Images were cropped, sized and arranged into panels using Adobe Photoshop CS version 8.0 (Adobe Systems).

Live-cell imaging HeLa cells stably expressing GFP–histone

H1.2 grown on 35-mm poly- L -lysine-coated glass-bottomed dishes (Matsunami) were transfected with siRNA sequences. The medium was changed to a CO 2 -independent medium (Gibco BRL) supplemented with 10% (v/v) FBS, 0.1 μg/ml penicillin/streptomycin, 20 mM glutamate and 100 mM Hepes before 1 h of imaging. The sequence of fluorescence images was acquired at every 6 min using a 40×, 1.4 NA oil objective on an inverted fluoresecence microscope (IX-81, Olympus) equipped with a z -motor and CCD (charge-coupled device) camera (Photometrics). Experiments were performed in a chamber maintained at 37 °C with a humidified atmosphere of 5% CO 2 in air. Metamorph software (Universal Imaging) and the WCIF (Wright Cell Imaging Facility) ImageJ Program ( http://www.uhnresearch.ca/facilities/wcif/fdownload.html ) were used for acquisition and analysis.

Online data Supplementary Figures S1-S3 Supplementary Movie 1 Supplementary Movie 2