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Supplementary Information for Materials and Methods
Cell culture, drugs, siRNA, and transfection
HeLa and 293T cells were cultured in DMEM supplemented with 10% v/v fetal bovine
serum. For synchronization in prometaphase, HeLa cells were treated for 18h with 2mM
thymidine, followed by multiple washes with PBS. After 4hr, cells were released in
fresh media and treated for 13hr with 200ng/ml nocodazole. Then the mitotic cells were
collected by shake-off and re-plated. When required,100ng/ml of cyclohexamide, 10M
of MG13, 100nM of Trichostatin A (TSA) or 100M of monastrol was added. Plasmid
and siRNA transfections were aided by Effectene (Qiagen) or LipofectamineTM 2000
(Invitrogen). Synthetic siRNAs were purchased from Samchully Pharmacy Company
(Korea), and the sequence information is as follows: BubR1-3’ directed against the 3’UTR region (5’-GTC TCA CAG ATT GCT GCC T-3’); siCDC20 #1 (GAA GAC CTG
CCG TTA CAT T); siCDC20 #2 (CAC CAG TGA TCG ACA CAT TC); siCdh1 (GAA
GGG UCU GUU CAC GUA UTT); siPCAF #1 (UCG CCG UGA AGA AAG
CGC A); siPCAF #2 (CCA CCA UGA GUG GUG UCU A); siAPC3#1 (GGA AAU
AGC CGA GAG GUA AUU); siAPC3#2 (CAA AAG AGC CUU AGU UUA AUU);
siMad2 (TCC GTT CAG TGA TCA GAC A).
Plasmids and antibodies
Various Myc-tagged BubR1 constructs were generated by PCR and subcloned into
pcDNA3.1-myc and pGEX 4T-1 vectors for expression in cultured cells and purification
from E. coli, respectively. The PCAF HAT domain was subcloned into pGEX 4T-1
(pGEX-PCAF-HAT) for purification from E. coli. For expression in cultured cells,
PCAF or CBP was subcloned into the pcDNA3XFLAG vector to produce
pCMV3XFLAG-PCAF and pcDNA3FLAG-CBP, respectively. pcDNA3 HA-p300 was
a generous gift of Dr. Chang Woo Lee (Sungkyungwan University, Korea). Various
BubR1 mutants such as K250R and K250Q were generated by site-directed mutagenesis
using pcDNA3.1-myc-BubR1 as the template. Each BubR1 mutant was then subcloned
into pDsRed express C1 (Clontech) or the adenoviral vector system, AdHTS viral vector.
The following antibodies were purchased: anti–Ac-K monoclonal, anti-GST (Upstate
Biotechnology); anti-Ac-K polyclonals (Cell Signaling); anti-PCAF (H-369), anti-CBP
(C-20), anti-Cdc20 (H-175), anti-Cdc27 (Sc-9972), anti-Mad2 (C-19), anti-Cyclin B (H433) and anti-CENP-E (H-300) (Santa Cruz); anti-β-tubulin (DM1A), anti-α-Actin (AC15) and anti-Flag (M2) (Sigma-Aldrich); anti-p300, anti-ubiquitin, and anti-BubR1 (BD
Biosciences); CREST (Cortex Biochem); anti-Aurora A (Trans Genic Inc.); anti-Plk1
(Zymed Laboratories); anti-Cdh1 (Calbiochem). Anti-Lamin A/C monoclonal
antibodies were a gift from Prof. F. McKeon (Harvard Medical School). The antipS676-BubR1 antibody was a gift from Dr. Erich A. Nigg (Max Planck Institute of
Biochemistry).
Immunoprecipitation and Western blot analysis
Cells were lysed in NETN buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris [pH 8.0],
0.5% NP-40) supplemented with protease inhibitors. Unless otherwise stated, 2.5 mg of
total cell lysate was subjected to immunoprecipitation (IP) with appropriate antibodies,
followed by SDS-PAGE and WB analysis.
Immunofluorescence microscopy
Indirect immunofluorescence microscopy was performed on cells grown on coverslips
and fixed in 4% paraformaldehyde. For β-tubulin staining, cells were fixed in cold
methanol at -20 °C and permeabilized twice in PBS-0.5% Triton X-100 (0.5% PBST)
for 15 min at room temperature (RT) before being processed for blocking and antibody
incubation, as described previously (Choi & Lee, 2008).
Chromosome spread for immunofluorescence assay (IFA)
To harvest prometaphase cells, HeLa cells were treated with 200 ng/ml of nocodazole
and subjected to mitotic shake-off. The cells were then incubated in 40% hypotonic
buffer (PBS diluted in tap water) for 5 min at RT and centrifuged at 1,000 rpm for 5 min
in a Cytospin (Shandon Cytospin4, Thermo Scientific). To remove cytoplasmic proteins,
slides were incubated in 0.1% Triton X-100 for 2 min and washed with PBS. After
fixation in 4% paraformaldehyde for 15 min at RT, the slides were incubated in 50 mM
NH4Cl for 5 min. Subsequently, they were subjected to immunofluorescence assay as
described above. Antibody dilutions were 1:100 for anti-BubR1 (mouse, BD
Biosciences) and 1:50 for anti-PCAF (H-369, SantaCruz).
Identification of acetylated lysine by Mass spectrometry
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was
performed using a delayed-extraction reflectron time-of-flight mass spectrometer
(Model MALDI-R; Micromass, UK). When analyzing acetylated lysine from
recombinant BubR1, BubR1 purified from insect cells were either subjected to in vitro
acetylation or left untreated; the two samples were then compared. For in vivo mass
spectrometry, HeLa cells were treated with nocodazole and subjected to mitotic shakeoff. Subsequently, the attached interphase cells and nocodazole-arrested cells were
subjected to immunoprecipitation with anti-BubR1 antibodies (BD Biosciences) and
SDS-PAGE. Coomassie-stained BubR1 bands were excised from interphase (attached)
and prometaphase (nocodazole-arrested and mitotic shake-off) cells and subjected to ingel digestion using pepsin or trypsin. MS/MS analysis was carried out by nanoflow
electrospray ionization (nano-ESI) on a Q-TOF2 mass spectrometer (Micromass, UK).
Acetylated peptides were identified by matching peptide masses from MALDI-TOF MS
with theoretical peptides derived from proteins in the NCBI database using the
MASCOT and Profound software packages.
Purification of recombinant BubR1 and in vitro acetylation assay
Full-length BubR1 was Myc-tagged at its N-terminus and subcloned into
pFastBacTMHT-B (Invitrogen), which codes for a 6 X-Histidine tag, to aid purification.
Virus production, infection, and affinity purification were performed according to the
manufacturer’s instructions. Purified BubR1 (0.5 and 1 ㎍) was analyzed by SDSPAGE to ensure that the protein employed in the in vitro acetylation assay was intact.
For the purification of proteins from E. coli, BubR1 (amino acids 1–514) and PCAF
(amino acids 352–832) were subcloned into pGEX 4T-1 (Amersham) and purified as
GST-fusion proteins. In addition, the BubR1 deletion mutant ∆BR1~5 was subcloned
into pGEX 4T-1. Combinations of the recombinant proteins indicated were incubated
for 1 hr at 30 °C in HAT buffer (250 mM Tris-HCl [pH 8.0], 50 % glycerol, 0.5 mM
EDTA, 5 mM dithiothreitol, and 1 mM unlabeled acetyl-coenzyme A or 0.05 Ci of [114
C] Acetyl-CoA). Reactions were run on SDS-PAGE and analyzed either by WB using
an anti-acetyl lysine antibody or by detection of radioactivity after gel drying, using a
BAS 2500 phosphor Imager (Fujifilm).
MPM2 and PI staining and mitotic index measurement
To determine the mitotic index, fixed cells were incubated for 1 h at RT with antiMPM2 antibodies (Upstate) diluted in PBS, 0.02% Triton X-100, and 2% BCS. Cells
were washed twice with PBS and incubated for 1 h at RT with Alexa Fluor 488 goatanti-mouse (Molecular Probes). Cells were treated with RNase, DNA was stained with
propidium iodide, and cells were analyzed on a FACS Canto (Becton Dickinson).
Time-lapse microscopy and fluorescence recording
HeLa cells stably expressing histone H2B-GFP were a generous gift from Dr. Toru
Hirota (The Cancer Institute, Japanese Foundation for Cancer Research). These cells
were grown in Delta-T dishes (Biotechs Inc., Butler, PA) for time-lapse fluorescence
and DIC microscopy. Images were taken and processed using a CoolSnap HQ-cooled
CCD camera on a DeltaVision Spectris Restoration microscope built around an
Olympus IX70 stand with a 40x/0.75 NA lens (AppliedPrecision). Cells expressing both
histone H2B-GFP and DsRed-BubR1 were tracked for 16 h, and images were collected
every 5 min. Quantification of BubR1 intensities (DsRed) was performed using Image J
software.
Adenovirus Infection
Adenovirus for BubR1, K250R, or K250Q was generated from NEWGEX Inc. (Seoul,
Korea). HeLa cells were infected with 100 MOI of GFP-fused WT-, K250R-, and
K250Q -BubR1 and GFP Adenovirus (as a control).