Supplementary Information
Materials and methods
Cell culture, reagents and tissue samples
SW48, DLD-1, HCT 116, and HT-29 (ATCC) were grown in DMEM with 10% FBS.
COLO 201 (ATCC) was grown in DMEM with 10% FBS and 1 mM sodium pyruvate.
Caco-2, LS 174T and CCD-18Co (ATCC) were grown in MEM with 10% FBS and 1
mM sodium pyruvate. K-562 (ATCC) was grown in DMEM with 10% FBS and 3 mM
sodium pyruvate. FHC (ATCC) was grown in the growth medium according to ATCC.
Polyclonal antibodies including anti-CHK antibody Lsk (C-20), anti-Src antibody Src2,
normal polyclonal IgG and anti-Csk (C-20) were from Santa Cruz. Polyclonal anti-CHK
antibodies CHK (C89) and Matk (N20) were raised against synthetic peptides and
purified using peptide-affinity columns (the epitope sequences are listed in
supplementary Figure 2). Anti-Src monoclonal antibodies MAb2-17 and MAb327 were
from Quality Biotechnology and from a hybridoma provided by Dr. Joan Brugge,
respectively. Polyclonal phospho-Src antibodies anti-Y530-P and anti-Y419-P were from
Cell Signaling. Monoclonal anti-tubulin antibody (Ab-1) was from Oncogene Sciences.
Monoclonal anti-GAPDH antibody was from IMGENEX. Human colon cancer tissues
and adjacent normal colon tissues from 6 patients were obtained from Alberta Research
Tumor Bank and the quality of cancers were further verified by histopathological
examination of the samples.
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Cell lysis in RIPA lysis buffer and western blotting
Subconfluent cells in tissue culture dishes were rinsed twice with cold PBS. Cells were
then lysed on ice for 10 min in lysis buffer, which is RIPA buffer (50 mM Tris-HCl, pH
7.2, 0.15 M NaCl, 1.0 mM EDTA, 0.1% SDS, 1.0% Triton X-100, 1.0% sodium
deoxycholate) freshly supplemented with phosphatase and protease inhibitors (1 mM
sodium orthovanadate, 7.5 mg/ml p-nitrophenolphosphate, 50 g/ml leupeptin, and 10
g/ml aprotinin). After 20 passes through 21-gauge needles, lysates were clarified by
centrifugation at 10,000 g for 20 min. Proteins were quantified using the Bradford assay,
and lysates were either used immediately or stored at -80°C. Colon tissues were rinsed
with PBS and homogenized in RIPA lysis buffer on ice using a Dounce Homogenizer and
clarified by centrifugation.
Proteins were resolved by SDS-PAGE and transferred to membranes. Antibodies
for CHK included CHK (C89) (1 g/ml), Matk (N20) (2 g/ml) and Lsk (C-20) (1
g/ml). Anti-Csk (1:200), anti-tubulin (0.5 g/ml), MAb327 (1 g/ml), Src2 (1 g/ml),
anti-Y419-P (1:1000) and anti-Y530-P (1:1000) were also used for western blotting.
Proteins were visualized by exposure to film following detection using enhanced
chemiluminescence (ECL Plus, Amersham), according to manufacturer’s instructions.
Quantification of protein bands was done by using ImageQuant software on a Storm 860
phosphorImager from Molecular Dynamics.
RT-PCR
Total RNA from mouse tissues (stabilized in RNAlater reagent, QIAGEN) and cultured
cells were isolated using RNeasy (QIAGEN) with individual protocols for brain, muscle,
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and tissues, and cultured cells respectively. PAGE-purified primer sets used to amplify
mouse CHK splicing variants 1 and 2 were as follows (Figure 2a): sense, 5'CCTCCGAGGGTCGCGCCTAAGCAG-3';
antisense,
5'-
AGAGTCAGATGCTGCAGGTCGAGTA-3', with predicted products of 650 bp and 798
bp
respectively.
For
splicing
variant
3,
the
sense
primer
was
5'-
CAAAGAGCCGCAGCATCTGACC-3', with a predicted product of 834 bp. Primers for
all human CHK splicing variants were as follows (Figure 3c): sense, 5'TACCGCGTCAAGCACCACACCAG-3';
CACTCTCCACTCTCTCGGTCCTCTG-3'.
antisense,
PCR
products
5'were
analyzed
electrophoretically and the ethidium bromide stained gels were photographed under UV
light.
Cell lysis in NP-40 lysis buffer, Src kinase assay and co-immunoprecipitation
Cells were lysed as in the RIPA lysis procedures except replacing RIPA lysis buffer with
NP-40 lysis buffer (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1.0 mM EDTA, 1% NP-40)
freshly supplemented with phosphatase and protease inhibitors, and lysing for 30 min on
ice with vortexing at 10 minute intervals.
Cell lysates were incubated with an excess of Src antibody MAb327 (0.5 g
antibody/100 g cell extract) for 1 h on ice, followed by addition of 30 l of 25% protein
G-agarose and 200 l NP-40 lysis buffer and rotation at 4 °C for 1 h. Mouse IgG was
used as control. The immune complexes were washed with NP-40 lysis buffer and with
kinase assay buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM DTT, 5 mM MgCl 2,
200 M sodium orthovanadate and 4 mg/ml p-nitrophenolphosphate). Src kinase activity
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was measured in the immmunoprecipitates by the addition of 50 l of kinase assay buffer
containing 0.02 mM ATP, 10 Ci of [-32P] ATP (3000 Ci/mmol), and 100 M Src
optimal peptide. Following incubation at 30 °C for 30 min, 25 l of 50% (v/v) acetic acid
was added to each tube, after which 50 l was spotted onto P81 phosphocelluose paper.
The filter papers were then washed four times with 0.5% phosphoric acid to remove free
ATP, rinsed once with acetone, dried, and counted in a scintillation counter.
Src immunoprecipitates described above, as well as CHK immunoprecipitates
using Lsk (C-20), were also analyzed by western blotting to examine coimmunoprecipitation of CHK and Src.
DNA Transfection
50% confluent DLD-1 cells were transiently transfected with pcDNA3 (Invitrogen),
pcDNA3-CHK (Chong et al., 2004) or EGFP C1 (Clontech) plasmid using
Lipofectamine 2000 (Invitrogen) as per the manufacturer’s instructions except replacing
Opti-MEM I Reduced Serum Medium with DMEM. 24 h later, cells were lysed with NP40 lysis buffer and subjected to western blotting, immunoprecipitation and Src kinase
assay.
Transfection of siRNA
siRNA duplexes of human CHK (sense, 5'-GCACCCAGUGUAUCACCAAdTdT-3';
antisense, 5'-UUGGUGAUACACUGGGUGCdTdT-3') and human Csk (sense, 5'GCAUCAUCCCAGCCAACUAdTdT-3';
antisense,
5'-
UAGUUGGCUGGGAUGAUGCdTdT-3') were designed using BLOCK-iT RNA
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Designer program (Invitrogen) as the top-rank siRNA sequences, and then the singlestranded sequences were synthesized in the RNA facility of University of Calgary.
siRNA duplexes of negative control (sense, 5'-UUCUCCGAACGUGUCACGUdTdT-3';
antisense, 5'-ACGUGACACGUUCGGAGAAdTdT-3') were designed by QIAGEN, and
the single-stranded sequences were synthesized in the RNA facility of University of
Calgary. Alexa488-labelled control siRNA duplex was from QIAGEN. Double-stranded
siRNA was prepared by heating the complementary single strands together at 90ºC for 1
min, then 37ºC for 1 h.
Cells grown in dishes at approximately 50% confluence were transiently
transfected with siRNA (CHK, Csk, control, or Alexa488-control), using siLentFect lipid
reagent (BIO-RAD) as per the manufacturer’s instructions. Transfections and siRNA
concentrations are: 1, Control siRNA (20 nM); 2, CHK siRNA (20 nM); 3, Csk siRNA
(20 nM); 4, CHK siRNA (10 nM) + Csk siRNA (10 nM). Cells were lysed 36 h later
using NP-40 lysis buffer for western blotting and Src kinase assay.
Immunofluresence
Cells grown on coverslips were fixed in formaldehyde and permeabilized (each step was
10 min at room temperature, sequentially in PBS containing 3.7% formaldehyde, 0.5%
Triton X-100, and 0.05% Tween 20), then incubated with 1 g/ml antibody (Lsk (C-20),
Mab327) in PBS containing 1% BSA for 45 min at 37ºC in a humidified chamber. After
washing 5 times in PBS containing 0.05% Tween 20, the cells were incubated with
secondary antibody for 45 min at 37ºC in the chamber. 2mg/ml Alexa Fluor488 chicken
anti-mouse IgG (H+L) from Molecular Probes (Eugene, OR) was diluted 500 fold, and
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1.5mg/ml Cy3-conjugated AffiniPure Donkey Anti-Rabbit IgG (H+L) from Jackson
Immunoresearch Laboratories (West Grove, PA) was diluted 100 fold in PBS containing
1% BSA. After immunostaining, the coverslips were washed 5 times with PBS, stained
with DAPI (Sigma, 1 g/ml in PBS), washed again and mounted with antifade reagent
(Molecular Probes). Specimens were observed and digital images were acquired with a
Zeiss-Axioskop microscope. Images were deconvoluted using ImageJ software.
Soft agar colony assay
2 × 103 trypsinized and separated DLD-1 cells were added to 3 ml of DMEM medium
containing 10% FBS, antibiotics, and 0.3% agarose. After vortexing briefly, the cells
were plated on 60 mm dishes over a 3 ml bottom layer of pre-hardened 0.5% agarose
medium. Additional 0.3% agarose medium was added every 3 days. The cells were
maintained at 37°C in a 5% CO2 incubator for 10 days and the colonies containing over
100 cells were counted with an inverted microscope.
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