Lind et al, Supplementary Information
Supplementary Information
Material
Cell lines
In the present study 20 colon cancer cell lines were included, counting nine with
microsatellite instability (MSI; Co115, HCT15, HCT116, LoVo, LS174T, RKO,
SW48, TC7, and TC71) and 11 microsatellite stable ones (MSS; ALA, Colo320, EB,
FRI, HT29, IS1, IS2, IS3, LS1034, SW480, and V9P). CRL-1790 and hTERT RPE-1
(epithelial lines from normal colon and retina, respectively) were also included. In
addition 30 cancer cell lines from other tissues were analyzed in the present study,
including breast (BT20, BT-474, Hs578, SK-BR-3, T47D, ZR-75-1, ZR-75-30),
cervix (HeLa), gastric (AGS, KATO III, NCI-N87), kidney (786-O, ACHN, Caki-1,
Caki-2), ovary (ES-2, OV-90, OVCAR-3, SK-OV-3), pancreas (AsPC-1, BxPC-3,
CFPAC-1, HPAF-II, PaCa-2, Panc-1), prostate (LNCaP), and uterus (AN3CA, HEC1-A, KLE, RL95-2). All commercially available cell lines have been purchased from
the American Type Culture Collection (ATCC, LGC Standards, Middlesex, UK). The
remaining cell lines have been obtained from collaborators. The breast cancer and
pancreatic cancer cell lines were kindly provided by Dr. Anne Kallioniemi, Tampere
University Hospital, Finland. Non-commercially available colon cancer cell lines
were kindly provided by Dr. Richard Hammelin, INSERM, Paris, France. None of the
cell lines have been authenticated. However, all colon cancer cell lines have
previously been extensively profiled on the genome level combining karyotyping (Gband analysis), comparative genomic hybridization (CGH), and multicolor
fluorescence in situ hybridization (M-FISH) (Kleivi et al. 2004). ACHN, AGS,
AN3CA, BT20, Caki-1, Caki-2, CRL-1790, ES-2, HEC-1-A, Hs578, KATO III, KLE,
LNCaP, NCI-N87, OV-90, RL95-2, T47D, and 786-O have been purchased directly
1
Lind et al, Supplementary Information
from ATCC in the period between 2005 and 2010 and have since then been cultured
only few passages. Standard culturing conditions were used and will be given on
request. For DNA and RNA extraction we used phenol and chloroform, and Trizol,
respectively.
Normal colorectal mucosa samples
The test series comprises 51 normal mucosa samples from 48 deceased colorectal
cancer free individuals (autopsy material collected at the Institute of Forensic
Medicine, Rikshospitalet, Oslo University Hospital; Supplementary table 1). Twentyseven of the samples were from the distal part and 24 from the proximal part of the
colorectum. The age of the individuals ranged from 22 to 86 years with a median
value of 55 years. The validation series comprises 59 normal mucosa sample biopsies
from 59 individuals attending a population-based sigmoidoscopy screening study
(Telemark, Norway; Supplementary table 1), harboring neither colorectal adenomas
nor carcinomas (Thiis-Evensen et al. 1999). The age of the individuals range from 63
to 72 years with a median value of 67 years. In addition, 105 normal mucosa samples
taken in distance from the carcinoma were included from the 105 patients in the
verification (carcinoma) series.
Stool samples
Paired colorectal carcinoma and stool samples from nine patients were analyzed,
including a stool sample taken orally from the colorectal carcinoma. All stool samples
were collected from the resected speciment post surgery and approximately two grams
(two table spoons) were added to a 50ml tube containing 20ml DNA stabilizing buffer
(0.5 mol/L Tris, 10mmol/L NaCl, 100mmol/L EDTA; pH7) and frozen at -80°C. Prior
2
Lind et al, Supplementary Information
to DNA extraction the sample was thawed and homogenized by vortexing and 800µl
homogenized stool sample was the input amount used in the QIAamp DNA Stool
Mini Kit (Qiagen). DNA was extracted according to the manufacturers’ protocol with
the following modifications: the volume of the lysis buffer was reduced to 800µl, and
the DNA was eluted in 50µl elution buffer. Three DNA solutions were extracted from
each stool sample and individually subjected to bisulfite treatment.
Methods
Bisulfite treatment of DNA
Prior to methylation analyses 1.3μg DNA from each tissue sample was bisulfite
modified using the EpiTect bisulfite kit (Qiagen Inc., Valencia, CA). For stool, three
individually extracted DNA solutions were bisulfite treated per sample, using an input
amount of 20μl.
Qualitative methylation-specific polymerase chain reaction (MSP)
MSP primers were designed using Methyl Primer Express v1.0 (Applied Biosystems,
Foster City, CA, USA) and purchased from MedProbe (Medprobe, Oslo, Norway).
They amplified fragments of 144 and 146 bases for the methylated and unmethylated
fragment, respectively. Both fragments covered the annotated transcription start site
of SPG20 (UCSC Genome Browser (Kent et al. 2002)). Primer sequences and general
information are listed in Supplementary table 2.
The MSP was carried out in a total volume of 25 ul containing 1 x PCR Buffer
(including 15 mM MgCl2; Qiagen), 200 μM of each dNTP (Amersham Biosciences,
Piscataway, NJ, USA), 800 pM of each primer (MedProbe), and one U HotStarTaq
3
Lind et al, Supplementary Information
DNA polymerase (Qiagen). Human placental DNA (Sigma-Aldrich) treated in vitro
with Sss1 methyltransferase (New England Biolabs, Ipswich, MA, USA) was used as
a positive control for the methylated MSP reaction, whereas DNA from normal
lymphocytes was used as a positive control for the unmethylated MSP. Water was
used as a negative control in both reactions. PCR products were mixed with five μl
gel loading buffer (1 x TAE buffer, 20% Ficoll; Sigma Aldrich, and 0.1% xylen
cyanol; Sigma Aldrich) and resolved by electrophoresis using 2% agarose (BioRad,
Hercules, CA, USA) in 1xTAE and ethidium bromide (Sigma Aldrich). Gels were
visualized by UV irradiation using a Gene Genius (Syngene, Frederick, MD, USA).
All results were confirmed by a second independent round of MSP and scored
independently by two authors (SAD and GEL). In cases with diverging results from
the two rounds of MSP and/or discrepancy in the scoring by the two authors, a third
run of MSP was performed. Representative MSP products from the methylated and
unmethylated reactions were sequenced in order to verify the identity of the amplified
product.
Quantitative methylation-specific polymerase chain reaction (qMSP)
qMSP primers and probe were designed using Primer Express v3.0 (Applied
Biosystems). Primers were purchased from MedProbe and the probe (labeled by 6FAM and a minor groove binder non-fluorescent quencher) was purchased from
Applied Biosystems. SPG20 qMSP primers amplified an 84 bp long fragment
overlapping with the MSP fragment (Supplementary figure S1).
The qMSP was performed in a 20μl reaction volume containing 0.9 μM forward and
reverse primers, 0.2 μM probe, 30 ng bisulfite treated template (tissue samples) or 1μl
4
Lind et al, Supplementary Information
bisulfite treated template (stool sample), and 1x TaqMan Universal PCR master mix
NoAmpErase UNG (including AmpliTaq Gold DNA polymerase and passive
reference; ROX) using the following PCR program: 95ºC for 10 minutes, then 45
cycles of 95ºC for 15 seconds followed by 60ºC for 1 minute. Each sample was
analyzed in triplicate (altogether nine replicates for each stool sample) in 384-well
plates using the 7900HT Sequence Detection System (Applied Biosystems), and the
median value was used for data analysis. A standard curve was generated from 1:5
serial dilutions of bisulfite-converted commercially available methylated DNA
(CpGenome Universal Methylated DNA; Millipore Billerica, MA, USA). The
commercially available methylated DNA sample was also used as a positive control
for the qMSP reaction. Additionally, all plates contained multiple water blanks,
bisulfite modified DNA from normal lymphocytes as well as unmodified DNA as
negative controls. An internal reference set directed against ALU sequences depleted
of CpG dinucleotides were included in the analysis to normalize for input DNA. This
reaction (ALU-C4) has previously been shown to be less susceptible to normalization
errors caused by cancer-associated aneuploidy and copy number changes
(Weisenberger et al. 2005). For all samples the level of methylated DNA (percent of
methylated reference, PMR) was calculated using the following formula:
[(SPG20/ALU)sample / (SPG20/ALU)positive control] x 100. For binominal analyses a fixed
threshold of 7.0 was used to categorize tissue samples as methylation positive (equal
or higher values) or negative (lower value). The threshold represented the percentile
of the highest PMR value obtained across the normal samples in the test series. With
the exception of an outlier with PMR 29, all normal samples in this series were
subsequently scored as metylation negative, ensuring a high specificity. For tissue
samples, amplification after cycle 35 was scored as negative (receiving a quantity of
5
Lind et al, Supplementary Information
0), according to the Applied Biosystems protocol recommendations. For stool samples
three parallel DNA solutions were isolated and bisulfite treated individually. Each
parallel was analyzed in triplicate in the qMSP reaction. Ct values equal to or higher
than that of the bisulfite treated normal blood control included in the same plate were
scored as negative (receiving a quantity of 0). A stool sample was scored as
methylation positive when a minimum of one out of the three sample parallels had a
positive PMR value. qMSP primer and probe sequences can be found in
Supplementary table 2.
Bisulfite sequencing
The initial PCR was carried out in a total volume of 25 μl containing 1 x PCR Buffer
(including 15 mM MgCl2; Qiagen), 200 μM of each dNTP (Amersham), 800 pM each
of forward and reverse bisulfite sequencing primer, and one U HotStarTaq DNA
polymerase (Qiagen). Excess primers and nucleotides were removed by ExoSAP-IT
treatment (GE Healthcare, Buckinghamshire, UK). One point five μl ExoSAP-IT
solution was added to 10 μl sample and incubated for 15 minutes at 37ºC and 15
minutes at 80ºC. Two μl of the purified product was added to a 10 μl sequencing
reaction containing 40 pM forward or reverse bisulfite sequencing primer, two μl
dGTP BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied
Biosystems), and 1 x BigDye Terminator v1.1 Sequencing Buffer. The sequencing
reaction products were purified using Sephadex G-50 Superfine powder (GE Health
Care) and sequenced in a 3730 DNA Analyzer (Applied Biosystems). The
approximate amount of methyl cytosine of each CpG site was calculated by
comparing the peak height of the cytosine signal with the sum of the cytosine and
thymine peak height signals, as previously described (Melki et al. 1999). CpG sites
6
Lind et al, Supplementary Information
with ratios ranging from 0 - 0.20 were classified as unmethylated, CpG sites within
the range 0.21 – 0.80 were classified as partially methylated, and CpG sites ranging
from 0.81 - 1.0 were classified as hypermethylated.
7
Lind et al, Supplementary Information
Supplementary figure legends
Supplementary Figure S1. Bisulfite sequencing confirmed methylation status as
assessed by methylation-specific polymerase chain reaction for SPG20.
The upper part is a schematic presentation of the CpG sites (vertical bars) amplified
by the bisulfite sequencing primers (-258 to 249, NM_015087). The transcription start
site is represented by +1 and the arrows indicate the location of the MSP and qMSP
primers. For the lower part of the figure, black circles represent methylated CpGs,
white circles represent unmethylated CpGs, and grey circles represent partially
methylated sites. The column of U, M, and U/M at the right side of this lower part
lists the methylation status of the respective cell lines as assessed by us using MSP
analyses. Abbreviations: MSP, methylation-specific polymerase chain reaction; s,
sense; as, antisense; p, probe; U, unmethylated; M, methylated; U/M, presence of both
unmethylated and methylated band; qMSP, quantitative methylation-specific
polymerase chain reaction.
Supplementary Figure S2. Endogenous Spartin partially co-localizes with tubulin.
HeLa cells were permeabilized with 0.05% Saponin prior to PFA fixation, to visualize
microtubule using confocal immunofluorescence microscopy. The cells were stained
with antibodies against Spartin (red) and -tubulin (green). Nuclei are visualized in
blue. Yellow in the merged pictures (B, C) indicate co-localization. C-E show
magnifications of the boxed region in A and B. Size bar 5um.
Supplementary Figure S3. Localization of Spartin in hTERT RPE-1 cells.
8
Lind et al, Supplementary Information
3D reconstructions of immunofluorescence confocal z-stack images showing the
localization of endogenous Spartin (red) and a-tubulin (green) in hTERT RPE-1 cells.
(A,B) show localization to the cytokinesis bridge. (C) Shows localization to the
spindle poles of prometaphase. Nuclei are shown in blue. The cells were
permeabilized with 0.05% Saponin prior to PFA fixation. Size bars 10 um.
Supplementary Figure S4. Localization of Spartin in prometaphase.
Confocal immunofluorescence images showing the difference in level of Spartin (red)
at the spindle poles of the cell lines indicated. -tubulin (green) DNA (blue). For
comparison, the pictures are generated with identical settings on the microscope. Note
the very intense staining of Spartin at the spindle poles of HeLa and CRL-1790 cells
as compared to the colon cancer cell lines. One of the spindle poles in the CRL-1790
cell is outside the confocal section. Trace amounts of Spartin, could occasionally be
detected at the spindle pools of the colon cancer cell lines. The cells were
permeabilized with 0.05% Saponin prior to PFA fixation. Size bar, 5um.
Supplementary Figure S5. SW480 cells have curved cytokinesis bridges and a
broad distribution of aurora B along the bridge.
Cytokinesis profiles of PFA fixed CRL-1790 cells (A) or SW480 cells (B) as seen
from above in 3D reconstructions of confocal z-stack images, stained for aurora B
(red), -tubulin (green) and DNA (blue). C and D show the respective cells in a side
view. Note the curved shape of the cytokinesis bridge between the dividing SW480
cells. E and F show how the Aurora B staining spreads out along the bridge in SW480
cells as compared to the normal colon epithelial cell line.
9
Lind et al, Supplementary Information
Supplementary Figure S6. Spartin is absent from cytokinesis bridges in SW480
cells.
SW480 cells were permeabilized with 0.05% Saponin prior to PFA fixation and
stained with antibodies against (A) -tubulin (green) and (B) Spartin (red). Arrows
point at examples of cytokinesis bridges devoid of Spartin. Note that the cells
indicated by numbers are linked via -tubulin labelled cytokinesis bridges, like pearls
on a string. Spartin could also not be detected on cytokinesis bridges in the three other
cancer cell lines tested in this study (not shown).
Supplementary Figure S7. Example of dividing SW480 cells having lagging
chromosomes in the enveloped cytokinesis bridge.
A) Cells were permeabilized with 0.05% Saponin prior to PFA fixation and stained
for α-tubulin (green) and DNA (blue Hoechst). B) Grey scale image of the DNA stain
in A. Note the DNA lining the cytokinesis bridge.
Supplementary Figure S8. Level of Spartin in control and Spartin depleted HeLa
cells.
Control or siRNA treated cells were fixed in methanol and stained for confocal
immunofluorescence analysis with anti Spartin (red) and anti -tubulin (green)
antibodies. Nuclei are visualized in blue. Note that the intensity of the Spartin signal
is significantly weaker in the knockdown cells compared to the control cells. A-C:
Interphase, D-I: Metaphase. The pictures are taken with identical intensity settings on
the microscope, to allow comparison of the Spartin signal. Size bar in A, 20um. Size
bar in D, 5um.
10
Lind et al, Supplementary Information
Supplementary Figure S9. Spartin is a regulator of late cytokinesis in hTERT
RPE-1 cells.
A) Western blot analysis of control and Spartin depleted hTERT RPE-1 cells, using
two independent siRNA duplexes. Two days post transfection. B) After siRNA
treatment, the cells were stained for Aurora B, α-tubulin and DNA and analyzed by
microscopy. The graph shows the percentage of cells in late cytokinesis. siRNA
treated hTERT RPE-1 cells show arrest in late cytokinesis. Error bars show +/-SEM
of 3 independent experiments. In total approximately 1000 cells were analyzed for
each condition. C) Spartin depleted HeLa cells or hTERT RPE-1 cells show reduced
growth rate relative to control transfected cells. Error bars show +/-SEM of 3
independent experiments. In total approximately 3000-4000 cells were analyzed for
each control, set to 1.
Confocal immunofluorescence images showing late cytokinesis profiles of control (D)
and Spartin depleted (E) hTERT RPE-1 cells were fixed in 3% PFA and stained for
Aurora B (red), α-tubulin (green) and DNA (blue). Note the difference between the
perfect late cytokinesis bridge in the control cells and the convoluted bridges in the
siRNA treated cells.
Supplementary Figure S10. Spartin is a regulator of late cytokinesis in HeLa
cells
A) Western blot analysis of control and Spartin depleted cells, using two independent
siRNA duplexes. B) siRNA treated cells show arrest in late cytokinesis (oligo1:
P=0.0029; oligo2: P=0.001; Independent samples t-test). After siRNA treatment, the
cells were stained for Aurora B, α-tubulin and DNA and analyzed by microscopy. The
graph shows the percentage of cells in different stages of mitosis indicated on the x-
11
Lind et al, Supplementary Information
axis. There was no significant difference in the quantitations between various time
points of knockdown, so the results were pooled. Error bars show +/-SEM of 7
independent experiment for control RNA and oligo1, and 4 experiments for oligo2. In
total approximately 30 000 cells were analyzed for control or oligo1, and 17 000 cells
for oligo2. C-E) Confocal immunofluorescence images showing late cytokinesis
profiles of control (C) and Spartin depleted (D-E) HeLa cells PFA fixed and stained
for Aurora B (red), α-tubulin (green) and DNA (blue). Note the difference between
the perfect late cytokinesis bridge in the control cells and the convoluted bridges in
the siRNA treated cells. Spartin depleted cells show increased number of profiles with
Aurora B localizing to the length of the bridge, consistent with an arrest at a very late
stage of cytokinesis.
12
Lind et al, Supplementary Information
References
Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM and Haussler D. (2002).
Genome Res, 12, 996-1006.
Kleivi K, Teixeira MR, Eknaes M, Diep CB, Jakobsen KS, Hamelin R and Lothe RA. (2004). Cancer
Genet Cytogenet, 155, 119-131.
Melki JR, Vincent PC and Clark SJ. (1999). Cancer Res, 59, 3730-3740.
Thiis-Evensen E, Hoff GS, Sauar J, Langmark F, Majak BM and Vatn MH. (1999). Scand J
Gastroenterol, 34, 414-420.
Weisenberger DJ, Campan M, Long TI, Kim M, Woods C, Fiala E, Ehrlich M and Laird PW.
(2005). Nucleic Acids Res, 33, 6823-6836.
13