Supplemental Data S1

PHB2 Protects Sister-ChromatidCohesion in Mitosis

Hideaki Takata, Sachihiro Matsunaga,Akihiro Morimoto, Nan Ma, Daisuke Kurihara,

Rika Ono-Maniwa, Masatoshi Nakagawa,Takachika Azuma, Susumu Uchiyama,

and Kiichi Fukui

Supplemental Experimental Procedures

Cells and Transfection

HeLa cells were grown in Dulbecco’s modified Eagle’s medium

(DMEM; GIBCO BRL) supplemented with 10% fetal-bovine serum

(FBS; Equitech-Bio). For construction of HeLa cells transiently

expressing GFP-PHB2 and Myc-PHB2, a cDNA of PHB2 (IMAGE:

3452360; Open Biosystems) was inserted into the pIC113 vector

(kindly provided by Professor I.M. Cheeseman, Ludwig Institute for

Cancer Research) pc DNA 3.1/myc-HisA (Invitrogen). We trans-

fected HeLa cells with the GFP-PHB2-encoding plasmid by using

the FuGene6 reagent (Roche). A GFP-tagged expression vector for

H1.2 visualization was constructed as described previously [S1].

Antibodies

For generating a rabbit PHB2 polyclonal antibody, a full-length

PHB2 cDNA was inserted into the pDEST17 vector (Invitrogen).

His-tagged PHB2 was expressed in Escherichia coli, purified with

a fast-protein liquid chromatography system (GE Healthcare), and

used to immunize a rabbit. The resulting antibodies raised were

affinity purified with the antigen and used at a dilution of 1:1000.

The other primary antibodies used in this study were as follows:

anti-Scc1 rabbit polyclonal (kindly provided by Professor K. Yoko-

mori, University of California), used at 1:100; anti-Sgo1 rabbit

polyclonal (kindly provided by Prof. Y. Watanabe, The University of

Tokyo), used at 1:1000; anti-B56a mouse monoclonal (BD Transduc-

tion Laboratories), used at 1:100; anti-Bub1 mouse monoclonal

(MBL), used at 1:20; anti-BubR1 mouse monoclonal (BD Transduc-

tion Laboratories), used at 1:1000; anti-Mad2 rabbit polyclonal (Co-

vance), used at 1:100; anti-CENP-A mouse monoclonal (Abcam),

used at 1:50; anti-AIM1 (Aurora B) mouse monoclonal (BD Transduc-

tion Laboratories), used at 1:200; anti-CENP-E mouse monoclonal

(Abcam), used at 1:100; anti-CENP-F rabbit polyclonal (Novus Bio-

logicals), used at 1:2000; anti-a-tubulin mouse monoclonal (Calbio-

chem), used at 1:100; anti-g-tubulin rabbit (Sigma), used at 1:2000;

anti-HEC1 mouse monoclonal (Affinity BioReagents), used at

1:1000; anti-Plk1 mouse monoclonal (Zymed Laboratories), used

at 1:1000; anti-Cdc27 mouse monoclonal (Abcam), used at 1:2000;

anti-securin mouse monoclonal (Abcam), used at 1:50; anti-Cyclin

B1 rabbit (Sigma), used at 1:500; anti-phospho histone H3 (Thr3)

rabbit monoclonal (Upstate Biotechnology), used at 1:500; anti-

GFP rabbit polyclonal (Invitrogen), used at 1:300; and CREST (Cor-

tex Biochem), used at 1:1000.

RNAi and Rescue Experiments

The siRNAs used in this study were as follows: PHB2 siRNA-1, 50-GA

AUCGUAUCUAUCUCACATT-30; PHB2 siRNA-2, 50-CUGAACCCC

UCU UGGAUUAAGTT-30; Aurora B siRNA [S2]; Mad2 siRNA [S3];

Plk1 siRNA [S4]; and HEC1 siRNA [S5]. The siRNAs were chemically

synthesized, and HeLa cells were transfected with 120 nM of each

siRNA duplex with Lipofectamine 2000 (Invitrogen) according to

the manufacturer’s instructions. For control transfection, a control

siRNA duplex (50-UUCUCCGAACGU GUCACGUTT-30; QIAGEN)

was used. Cells were collected at 48 hr after transfection and sub-

jected to further analyses.

For siRNA rescue assay, three silent mutations were introduced

into Myc- or GFP-PHB2 vector, changing nucleotide sequences at

814–822 position of PHB2 to 50-TACCTGACC-30 (italic nucleotides

indicate silent mutations). The RNAi-refractory constructs were

transfected into HeLa cells with FuGENE6 (Rosh) at 4 hr before

transfection of siRNA.

Immunofluorescence Microscopy

HeLa cells grown on coverslips were fixed with 4% paraformalde-

hyde (PFA) in phosphate-buffered saline (PBS [pH 7.4]) for 15 min

at 37�C. Alternatively, for Mad2, CENP-F, and B56a staining, cells

were fixed with 100% ice-cold methanol for 10 min at 220�C. For ki-

netochores-fiber analysis, cells were incubated in a calcium-

containing buffer (100 mM PIPES [pH 6.8], 1 mM MgCl2, 0.1 mM

CaCl2, and 0.1% Triton X-100) for 2 min at 37�C to depolymerize

the nonkinetochore microtubules; this was followed by fixation in

the same buffer supplemented with 4% PFA. For depolymerization

of unstable kinetochore microtubules, cells were incubated in ice-

cold medium for 10 min prior to incubation in the calcium-containing

buffer. Fixed cells were permeabilized with 0.2% Triton X-100 in PBS

for 10 min at room temperature and blocked with 1% BSA-PBS for

15 min. Metaphase-chromosome spreads were prepared as de-

scribed previously [S6]. Next, the cells were incubated with primary

antibodies for 1 hr at room temperature, and this was followed by

incubation with Alexa 488- or TRITC-conjugated secondary anti-

bodies (1:200 dilution) for 1 hr. After washing, the cells were stained

with 1 mg/ml of 40,6-diamidino-2-phenylindole (DAPI) in Vectorshield

mounting medium (Vector Laboratories) and observed under an

Axioplan II imaging fluorescence microscope (Carl Zeiss) equipped

with a cooled CCD camera (MicroMax; Roper Scientific) driven by

the IP Lab software. We captured deconvoluted images with

a cooled CCD camera (CH 350/L; Roper Scientific) by using an IX-

70 microscope (Olympus) and analyzed them by using the Delta

vision software (Applied Precision).

Live-Cell Imaging

HeLa cells stably expressing GFP-histone H1.2 were cultured in

35 mm glass-bottom dishes (Matsunami) and transfected with

siRNAs with Lipofectamine 2000 (Invitrogen). The medium was

changed to a CO2-independent medium (GIBCO BRL) supple-

mented with 10% FBS, 0.1 mg/ml of penicillin/streptomycin,

20 mM glutamine, and 100 mM HEPES at 1 hr before imaging. The

dishes were placed on the inverted platform of a fluorescence micro-

scope (IX-81; Olympus) equipped with a CO2 chamber set at 37�C.

Fluorescence images were acquired every 3 min with a 403 objec-

tive controlled with the MetaMorph software (Universal Imaging

Corporation). Stacks of images were assembled and processed

with the MetaMorph software.

Fluorescent In Situ Hybridization

RP11-113F1 BAC clone in the tff1 locus on chromosome 21 was

obtained from BACPAC Resources in the Children’s Hospital and

Research Center (Oakland, CA). FISH probes were labeled with

a nick-translation kit (Roche) and digoxigenin-11-dUTP (Roche).

Human cot-1 DNA (Invitrogen) and salmon sperm DNA (Wako)

were added, and probes were precipitated and resuspended in

hybridization buffer (50% formamide, 10% dextrane sulfate, and

23 SSC). HeLa cells were synchronized by double-thymidine treat-

ment, and interphase nuclei were obtained according to the method

by Schmitz et al. [S7]. After transfection of control and PHB2 siRNAs

for 48 hr, cells were treated with trypsin and harvested with centrifu-

gation at 310 g. Cells were then fixed in methanol/acetic (3:1, vol/vol)

and dropped onto the glass slides. The glass slide was incubated at

37�C for 16 hr, denatured in 70% formamide/23 SSC at 78�C for

1.5 min, and dehydrated by passage through an ethanol series

(70%, 90%, and 100%). The labeled probes were denatured at

92�C for 10 min, cooled on ice, and then applied to the slides. The

slides with the probe were denatured at 80�C for 2 min and incu-

bated for 12 hr at 37�C in a humid chamber. The hybridized probe

was detected with the fluorescent antibody enhancer set for DIG

detection kit (Roche). DNA was 1 mg/ml of DAPI in Vectorshield

mounting medium (Vector Laboratories). Acquisition of deconvo-

luted images was performed as described above.

Supplemental References

S1. Gambe, A.E., Maniwa-Ono, R., Matsunaga, S., Kutsuna, N.,

Higaki, T., Higashi, T., Hasezawa, S., Uchiyama, S., and Fukui,

K. (2007). Development of a multistage classifier for a monitoring

system of cell activity based on imaging of chromosomal

dynamics. Cytometry 71A, 286–296.

S2. Ditchfield, C., Johnson, V.L., Tighe, A., Ellston, R., Haworth, C.,

Johnson, T., Mortlock, A., Keen, N., and Taylor, S.S. (2003).

Aurora B couples chromosome alignment with anaphase by

targeting BubR1, Mad2, and Cenp-E to kinetochores. J. Cell

Biol. 161, 267–280.

S3. Tang, Z., Sun, Y., Harley, S.E., Zou, H., and Yu, H. (2004). Human

Bub1 protects centromeric sister-chromatid cohesion through

Shugoshin during mitosis. Proc. Natl. Acad. Sci. USA 101,

18012–18017.

S4. Tang, Z., Shu, H., Qi, W., Mahmood, N.A., Mumby, M.C., and Yu,

H. (2006). PP2A is required for centromeric localization of Sgo1

and proper chromosome segregation. Dev. Cell 10, 575–585.

S5. Joseph, J., Liu, S.T., Jablonski, S.A., Yen, T.J., and Dasso, M.

(2004). The RanGAP1-RanBP2 complex is essential for microtu-

bule-kinetochore interactions in vivo. Curr. Biol. 14, 611–617.

S6. Ma, N., Matsunaga, S., Takata, H., Maniwa-Ono, R., Uchiyama,

S., and Fukui, K. (2007). Nucleolin functions in nucleolus forma-

tion and chromosome congression. J. Cell Sci. 120, 2091–2105.

S7. Schmitz, J., Watrin, E., Lenart, P., Mechtler, K., and Peters, J.M.

(2007). Sororin is required for stable binding of cohesin to chro-

matin and for sister chromatid cohesion in interphase. Curr. Biol.

17, 630–636.

S8. Kasashima, K., Ohta, E., Kagawa, Y., and Endo, H. (2006). Mito-

chondrial functions and estrogen receptor-dependent nuclear

translocation of pleiotropic human prohibitin 2. J. Biol. Chem.

281, 36401–36410.

Figure S1. Localization Analysis of PHB2 during Mitosis

(A) HeLa cells expressing GFP-PHB2 (green) were fixed with 4% PFA, and centromeres were detected by CREST (red). DNA was stained with

DAPI (blue). The exogenous GFP-PHB2 is localized in the cytoplasm and the chromosomes of prophase and prometaphase cells, whereas it is

mainly localized in the cytoplasm and spindles of metaphase and anaphase cells. The scale bar represents 5 mm.

(B) Deconvoluted images of the prophase cell expressing GFP-PHB2. HeLa cells expressing GFP-PHB2 were pre-extracted by 0.01% Triton

X-100 before 4% PFA fixation and immunostained with GFP antibody (green) and CREST (red). DNA was stained with DAPI (blue). The scale

bar represents 5 mm.

S2

Figure S2. RNAi of PHB2 Using siRNAs with Different Sequences

For the control siRNA, we used a random RNA sequence. For the PHB2 siRNA, two siRNAs (PHB2 siRNA-1 and PHB2 siRNA-2) with different

sequences were designed.

(A) Immunoblotting analysis revealed that the two siRNAs cause 70% and 90% reductions in the expression of PHB2, respectively.

(B) At 48 hr after transfection with each siRNA, HeLa cells were fixed with 4% PFA and stained with DAPI so that the chromosome morphologies

could be observed. Depletion of PHB2 with either siRNA causes similar aberrant chromosomes. However, when we used PHB2 siRNA-2, the

significant reduction in PHB2 decreased the cell viability, as previously reported [S8]. In fact, a further reduction in PHB2 after a long-term

incubation with PHB2 siRNA-1 also caused cell death. Therefore, we used PHB2 siRNA-1 for our experiments. The scale bar represents 10 mm.

(C) The percentage of defects in sister-chromatid cohesion. The defects in sister-chromatid cohesion observed in PHB2 RNAi cells expressing

Myc or GFP (w45%) are rescued by the expression of RNAi-refractory Myc- or GFP-PHB2 (w15%). n = 5, >100 cells were counted in each

experiment.

(D) The expression levels of exogenous PHB2 were confirmed by immunoblotting. By the transfection of PHB2 siRNA-1, the expressions of

endogenous PHB2 and exogenous Myc-PHB2 (WT; non-RNAi-resistant form) were significantly decreased. Similarly, RNAi-resistant GFP-

PHB2 also showed a significant decrease of its expression by PHB2 RNAi (data not shown). By contrast, the expression of exogenous RNAi-

resistant Myc-PHB2 did not show any significant decrease of its expression.

S3

Figure S3. Defects in Chromosome Dynamics in PHB2-Depleted Cells

(A) PHB2 was depleted in HeLa cells stably expressing GFP-H1.2. The number in each panel shows the time point (min) after nuclear-envelope

breakdown (NEBD). In PHB2-depleted cells, four types of aberrant chromosome behaviors (I–IV) are observed: I, chromosomes never form a dis-

tinct metaphase plate and are scattered throughout the cytoplasm; II, chromosomes are aligned at the metaphase plate but begin to scatter

throughout the cytoplasm after a long period of arrest at metaphase; III, chromosomes are segregated without alignment at the metaphase plate;

and IV, chromosomes are aligned at the metaphase plate, but the cells are arrested at metaphase for a long time. These aberrations are not

observed in control cells. The scale bar represents 5 mm.

(B) The time intervals between NEBD and the onset of anaphase were measured in movies of control and PHB2 RNAi cells. PHB2-depleted cells

show a significant delay in the onset of anaphase. Note that PHB2-depleted cells can be identified by their aberrant chromosome movement.

(C) Frequencies of the aberrations of chromosome dynamics in PHB2 RNAi cells. Categories I–IV correspond to the numerals indicated in (A).

S4

Figure S4. Mitotic-Spindle-Checkpoint Activity in PHB2 RNAi Cells

(A) The spindle-checkpoint activities in fixed control and PHB2 RNAi cells were determined by immunostaining for Bub1, BubR1, and Mad2

(green). The kinetochores and DNA were also stained with CREST (red) and DAPI (blue), respectively. Positive signals for spindle-checkpoint

proteins are detected in aberrant chromosomes in PHB2 RNAi cells, similar to control prometaphase cells. The scale bar represents 5 mm.

(B) Mitotic indexes after depletion of Aurora B or Mad2 in control and PHB2 RNAi cells. Knockdown of Aurora B or Mad2 suppresses mitotic

arrest in PHB2-depleted cells. n = 3; >1000 cells were counted in each experiment.

S5

Figure S5. Mitotic-Spindle Morphologies in PHB2 RNAi Cells

(A) Mitotic spindles were visualized by immunostaining for a-tubulin (green) and g-tubulin (red). DNA was stained with DAPI (blue). The scale bar

represents 5 mm.

(B) Three types of aberrant spindle morphologies, namely multipolar, elongated, and disorganized, are observed in PHB2-depleted cells.

(C) Frequencies of the spindle-aberration phenotypes shown in (B). Elongated mitotic spindles are frequently observed in PHB2-depleted cells.

n = 5; >1000 cells were counted in each experiment.

(D) Intercentrosome distances in control metaphase cells and PHB2-depleted mitotic cells. We determined the intercentrosome distance by

measuring the distance between the two g-tubulin signals in a cell; n = 10.

(E) Kinetochore microtubule stability in RNAi-treated cells. Control, PHB2, and Hec1 siRNA-transfected cells were cold-treated, processed for

immunostaining, and stained with an a-tubulin antibody (green) and DAPI (blue). The Hec1 siRNA was used as a control for destabilizing kinet-

ochores-microtubule formation. The fluorescence intensity of the kinetochore microtubules is decreased in PHB2-depleted cells, similar to that

of kinetochores microtubules in Hec1-depleted cells. The scale bar represents 5 mm.

S6

Figure S6. Localizations of Outer Kinetochore Proteins in PHB2-Depleted Cells

(A) CENP-E, CENP-F, and Hec1 (green) were visualized by immunostaining in control and PHB2 RNAi cells. The centromeres and DNA were

stained with CREST (red) and DAPI (blue), respectively. Scale bars represent 5 mm.

(B) Fluorescence intensities of the outer kinetochore proteins. After immunostaining for CENP-E, CENP-F, and Hec1, the fluorescence intensities

of the proteins in cells were measured with the MetaMorph software. After PHB2 depletion, the intensity of each outer kinetochore protein is

decreased to <50% of the level in control cells; n = 5.

S7

Figure S7. Sister-Chromatid Cohesion in

RNAi Cells during S/G2 Phase

(A) HeLa cells were synchronized by double-

thymidine treatment. Cells were harvested

5 hr after release from the second arrest.

FISH analysis with a probe specific for the

trisomic tff1 locus on chromosome 21 could

successfully detect three pairs of centromere

signals (green) both in control and PHB2

RNAi cells. DNA was counterstained with

DAPI (blue). The paired signals indicated by

white arrows were magnified (6003) in the

lower-left inset. When HeLa cells were har-

vested at 9 hr after release from the second

arrest, PHB2-depleted mitotic cells showed

chromosome aberrations. These results

indicated that PHB2 were depleted in most

of G2-phase nuclei observed in the FISH

experiment and showed defects in chromo-

some congression when they entered mito-

sis. The scale bar represents 5 mm.

(B) The distance between paired FISH signals

in RNAi cells. There was no significant differ-

ence in the interchromatid distance between

control (0.40 6 0.12 mm) and PHB2 RNAi cells

(0.36 6 0.16 mm); n > 20 per condition.

Figure S8. Percentages of Aberrant Chromosomes in the Presence

or Absence of MG132 in RNAi Cells

At 45 hr after siRNA transfection, HeLa cells were incubated with

20 mM MG132 for 3 hr to inhibit the anaphase transition. The percent-

ages of aberrant chromosomes in mitotic cells among either control

or PHB2 RNAi cells are not affected by the MG132 treatment. This

indicates that the aberrant chromosomes in PHB2-depleted cells

are generated before the onset of anaphase and that prolonged

metaphase cannot rescue the chromosome congression defects

in PHB2-depleted cells. n = 3; >1000 cells were counted in each

experiment.

S8

Figure S9. Immunostaining of Scc1 in Inter-

phase and Anaphase

(A) HeLa cells were pre-extracted by 0.5%

Triton X-100 and fixed with 4% PFA. Scc1

was detected by Scc1 antibody (green), and

centromeres were visualized by CREST.

There was no difference in Scc1 localization

of interphase nuclei between control and

PHB2 RNAi cells. This indicated that loading

of PHB2 into interphase nuclei occurred nor-

mally in PHB2-depleted cells. The scale bar

represents 5 mm.

(B) In control cells, Scc1 signals were ob-

served at the centromeric region in prometa-

phase. In anaphase, Scc1 signals disap-

peared. The scale bar represents 5 mm.

S9

Figure S10. Immunoblotting to Confirm the

Protein Expression Levels in RNAi Cells

(A) The efficiencies of double knockdown of

PHB2/Aurora B, PHB2/Mad2, and PHB2/

Plk1 were investigated by immunoblotting.

(B) The expression levels of the cell cohesin

subunit Scc1 and cycle-related proteins

CDC27, CDC20, and Plk1 do not change after

PHB2 RNAi. There are no differences in the

expression levels of any of the cell-cycle-

related proteins between control and PHB2

RNAi cells. These results indicate that the

cell-cycle arrest observed in PHB2-depleted

cells is caused by spindle-checkpoint activa-

tion rather than failure of metaphase exit.

S10