Supplementary data
Supplementary Table 1. The antibodies used and their sources
Antibody
anti-SGT1
Type
rabbit polyclonal
Source
K. Kitagawa
anti-SGT1(612104)
mouse monoclonal
anti-BUB1(MAB3610)
mouse monoclonal
anti-BUB1
sheep polyclonal
anti-BUBR1 (MAB3612)
mouse monoclonal
anti-BUBR1 (612503)
mouse monoclonal
anti-MAD2 (sc-6329)
goat polyclonal
anti-HSP90 (sc-12119)
mouse monoclonal
anti-HSC70 (sc-7298)
mouse monoclonal
anti-SKP1 (sc-7163)
rabbit polyclonal
anti-CDC37 (sc-132129)
mouse monoclonal
anti–CENP-B
rabbit polyclonal
anti–CENP-C
rabbit polyclonal
anti–CENP-E
mouse monoclonal
anti-CENP-E (sc-11291)
goat polyclonal
anti–CENP-H (612142/612143)
mouse monoclonal
anti-CENP-H (A400-007A)
rabbit polyclonal
anti–CENP-I
anti-HEC1 (NB-100-338)
rabbit polyclonal
mouse monoclonal
BD Transduction
Laboratories
Chemicon
International, Inc.;
Temecula, CA
a gift from S.S.
Taylor, University of
Manchester,
Manchester, UK
Chemicon
International, Inc.
BD Transduction
Laboratories; San
Jose, CA
Santa Cruz
Biotechnology, Inc.;
Santa Cruz, CA
Santa Cruz
Biotechnology, Inc.;
Santa Cruz, CA
Santa Cruz
Biotechnology, Inc.
Santa Cruz
Biotechnology, Inc.
Santa Cruz
Biotechnology, Inc.
a gift from W.C.
Earnshaw, University
of Edinburgh,
Institute of Cell and
Molecular Biology,
Edinburgh, UK
a gift from W.C.
Earnshaw
a gift from T.J. Yen,
Fox Chase Cancer
Center, Philadelphia,
PA
Santa Cruz
Biotechnology, Inc.
BD Transduction
Laboratories
Bethyl Laboratories
Inc.; Montgomery,
TX
a gift from T.J. Yen
Novus Biologicals,
Inc.; Littleton, CO
1
Reference
Niikura and Kitagawa,
2003
Johnson et al., 2003;
Taylor et al., 1998;
Taylor et al., 2001
Tomkiel et al., 1994
Tomkiel et al., 1994
Schaar et al., 1997
Liu et al., 2003
anti-survivin (SURV-11S)
mouse monoclonal
anti-AIK (Aurora A) (3092)
rabbit polyclonal
anti–AIM-1 (Aurora B)
(611082/611083)
anti–-tubulin (63781)
mouse monoclonal
anti–glyceraldehyde-3-phosphate
dehydrogenase (GAPDH)
anti-centromere autoimmune
(ACA) serum
mouse monoclonal
mouse monoclonal
human polyclonal (from
a patient with the
autoimmune disease
scleroderma CREST
syndrome)
Alpha Diagnostic
International, Inc.;
San Antonio, TX
Cell Signaling
Technology, Inc.;
Beverly, MA
BD Transduction
Laboratories
ICN Biomedicals,
Inc.; Irvine, CA
Chemicon
International, Inc.
a gift from W.R.
Brinkley, Baylor
College of Medicine,
Houston, TX
Earnshaw and
Rothfield, 1985;
Valdivia and
Brinkley, 1985; Van
Hooser et al., 2001
Supplementary Table 2. A summary of the effects of 17-AAG treatment (Figure 1e) and
use of HSP90 siRNA (Figure 2d) on the localization of kinetochore proteins.
Kinetochore protein
Mitotic phase
CENP-H
Prophase/Prometaphase
Metaphase
Interphase
Prophase/Prometaphase
Metaphase
Interphase
Prophase/Prometaphase
Metaphase
Interphase
Prophase/Prometaphase
Metaphase
Interphase
Prophase/Prometaphase
Metaphase
Interphase
CENP-I
BUB1
CENP-C
CENP-B
Remaining kinetochore signals (%)
+ 17-AAG
+ HSP90 siRNA
11
18
17
24
34
46
27
67
34
44
66
41
65
79
80
60
N.D.
N.D.
100
100
100
95
100
100
100
100
100
100
100
100
N.D., not determined.
2
Supplemental figure legends
Figure S1 Delocalization of kinetochore proteins in response to 17-AAG treatment.
Signal from the CREST antiserum (red) and DAPI staining (blue) are shown. After 17AAG treatment, (a) the BUBR1 signal (green) at kinetochores was diminished during
prophase and metaphase; (b) the CENP-E signal (green) at kinetochores was decreased
during prophase and metaphase; (c) localization of HEC1 (green) to kinetochores was
affected substantially during metaphase and slightly during prophase; (d) localization of
BUB1 (green) to kinetochores was affected moderately during metaphase and slightly
during prophase; and (e) localization of CENP-B (green) was not substantially affected.
(f) Western blot analysis of kinetochore proteins in untreated HeLa cells (−) and in HeLa
cells treated with 17-AAG (500 nM) for 24 h (+) showed that the total amount of each
kinetochore protein is not substantially changed by 17-AAG treatment.
Figure S2 Delocalization of kinetochore proteins in response to HSP90 siRNA treatment.
HeLa cells were transfected with siRNAs that targeted either luciferase (Luc) or
HSP90alpha and HSP90beta. (a) BUB1 was delocalized in response to HSP90 depletion
by specific siRNA in cells at prophase and metaphase. (b) CENP-B did not delocalize in
response to HSP90 depletion. (c) Simultaneous HSP90 depletion and 17-AAG treatment
in cells at prophase (blue bars), metaphase (red bars), or interphase (yellow bars) yielded
results similar to those observed after either treatment alone.
Figure S3 (a) The molecular masses (in Daltons) after deconvolution of the spectra are
shown.
3
(b) The peptide sequences identified by the mass spectrometric analysis of SGT1
immunoprecipitates and the percentage of the total sequence covered by the identified
sequences. (c) Immunoblot analyses of (left) SGT1 immunoprecipitates and (right)
HSP90 immunoprecipitates. Proteins in the cell lysate (input, IN), supernatant (S), and
precipitate (P) obtained by using antibodies to SGT1 or HSP90 or mouse IgG were
detected with antibodies to BUBR1, BUB1, HEC1, CENP-B, CENP-I, CENP-E, and
CENP-H. Total cell lysate volumes were equivalent to 5% or 10% of that of the starting
material.
Figure S4 Kinetochore ultrastructure. (A) Low-magnification electron micrograph of
chromosomes in untreated HeLa cells in metaphase. Scale bar = 1 μm. (B) Highermagnification view of the inset shown in A. Scale bar = 200 nm. (C) Metaphase
alignment in a HeLa cell treated with 17-AAG (as described in Figure 3b, lane C). Scale
bar = 1 μm. (D) Higher-magnification view of the inset shown in C. Scale bar = 200 nm.
Appendix 17-AAG induces a mitotic delay that depends on the spindle checkpoint. (a)
Mitotic arrest caused by 17-AAG was reduced by MAD2 siRNA. 17-AAG (500 nM) was
added 48 h after MAD2 siRNA transfection. Cells grown on coverslips were fixed,
stained with DAPI, and subjected to fluorescence microscopy. Percentages of cells in
prophase, prometaphase, and metaphase (Mitotic index) are shown. A total of 500 cells
were analyzed in three independent experiments. (b) Western blot analysis of total HeLa
cell lysates harvested 72 h after transfection with siRNA duplexes directed against
4
MAD2 showed depletion of MAD2 protein. Luciferase (Luc) siRNA was transfected into
the cells as a control, and beta-tubulin protein served as a control for loading.
5