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Spplemental materials
The information for detailed experimental procedures was included in this section.
Cells and DNA Transfection
HeLa (cervical carcinoma) and MCF-7 (breast adenocarcinoma) cells were obtained
from The Cell Bank of Tohoku University, Sendai, Japan, and WI-38 (fetus lung
fibroblast) cells from HSRRB, Osaka, Japan. HT-29 (colon adenocarcinoma) cells were
from ATCC (Manassas, VA). All were maintained in DMEM supplemented with 10%
fetal bovine serum in a humidified atmosphere containing 5% CO2. Normal human
fibroblasts (NHDF, Cambrex, Walkerville, MD) and keratinocytes (NHEK, Kurabo,
Osaka, Japan) were cultured in non-serum medium with supplements recommended by
respective suppliers. Sialidase expression vectors were constructed by subcloning
NEU3 cDNA into an expression vector pCAGGS (a generous gift of Dr. Miyazaki,
Osaka University School of Medicine). DNA transfection was accomplished transiently
using the Effectene reagent (QIAGEN).
Synthesis of siRNA and Transfection
siRNAs targeting NEU3 and non-targeting siRNAs (scrambled siRNA and non-specific
control duplex VIII) were obtained from Dharmacon RNA Technologies, Inc.
(La-fayette, CO). The sequences of siRNA candidates for NEU3 were evaluated
according to rational design criteria of the manufacturer’s protocol, and specificity of
the sequences was confirmed by BLAST searches against the human genome sequence.
Three siRNAs were selected from the top highest scored candidates by analyzing
specific silencing of the NEU3 gene as described below. The sequences selected were
siRNA1 (GTATACCTACTACATCCCT), siRNA 2 (GTGAAGGCTTTCAGAGACT)
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and siRNA 3 (AAGGGAGTGTGGTAAGTTT) beginning at nucleotides 534, 779 and
839, respectively, of the NEU3 ORF sequence, and the scrambled siRNA was Sc
(GCGATTAATGTAGGTTCGA). Transfection was performed in 60 mm dishes with
Lipofectamine 2000 (Invitrogen) for HeLa cells, MCF-7 cells, and WI-38 cells, and by
nucleofection with the Nucleofector System (Instrumentation Lab.) for HT-29 cells,
NHDF and NHEK cells. At 24-48 h after transfection, cells were used for
experimentation.
Quantitative Analysis of Transcripts by Real-Time PCR
Quantitative analysis of the transcripts for NEU3 and other apoptosis-related molecules
was performed by real-time PCR using a LightCycler rapid thermal cycler system
(Roche) with QuantiTect SYBR Green PCR master mix (Qiagen) as described
previously (Yamaguchi et al, 2005). Fluorescence from SYBR Green bound to the PCR
product was detected, and specificity of the reactions was confirmed by melting curve
analysis and subsequently by agarose gel electrophoresis. The sequence-specific primers
for NEU3 were 5’-GACTGGTCATCCCTGCGTAT-3’(forward), and
5’-GAGCCATGATTCTGA CGGTGTT-3’ (reverse), which yielded a 469-bp fragment.
The others were: for Bax, 5’-GCGAGTGTC TCAAGCGCATC-3’(forward) and
5’-CCAGTTGAA GTTGCCGTCAGAA-3’(reverse); Bcl-Xl,
5’-CTTGCAGTTCAGCACCACCCTA -3’(forward), and
5’-GTGAGGCAGCTGAGGCCATAA-3’ (reverse); melanoma differentiation
associated gene-7 (mda 7), 5’-ACCCACAGCTATGCCTCTGATTG -3’(forward), and
5’-TGTTAAATTGGCGAAA GCAGCTC -3’ (reverse); GM3 synthase,
5’-TTTGGAAGCAGGTGGCAGAA-3’ (forward) and 5’-GTGGCTAAGACAA
CGGCAATGA-3’(reverse) and Gb3 synthase,
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5’-ACCTGCGGAACCTGACCAAC-3’(forward) and
5’-CATGCACAGCGCCATGAAC-3’(reverse). To correct for differences in the RNA
quality and quantity between samples, glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was used as an internal control. To measure the mRNA levels of other types
of human sialidases, the primers were prepared as described previously (Yamaguchi et
al., 2005).
Sialidase Assays
Crude extracts were prepared and assayed sialidase activity using GM3 (Alexis
Biochemicals, Lansen, Switzerland) as the substrate with Triton X-100 as described
elsewhere (Miyagi et al., 1999). After incubation, the amount of sialic acid released was
determined by fluorometric high -performance liquid chromatography with
malononitrile (Li, 1992). One unit of sialidase was defined as the amount of enzyme
catalyzed the release of 1 nmol of sialic acid/h.
Immunoblotting and Ras Activation Assays
Cells were homogenized and solubilized in 5 volumes of ice-cold buffer (50 mM Hepes
pH7.5, 150 mM NaCl, 1% NP-40, 0.25% deoxycholate, 2mMEDTA, 10 mM NaF, 2
mM Na3VO4, 2 mM PMSF, 7.5 g/ml aprotinin, and 10 g/ml leupeptin) for
immunoprecipitation or immunoblotting. The cell lysates or immunoprecipitates were
separated on SDS-PAGE under reducing conditions, and analyzed by immunoblotting
with the respective antibodies using ECLPlus Western blotting reagent (Amersham
Biosciences). The antibodies to phoshotyrosine (PY20) and phosphoserine were
obtained from BD Biosciences (San Jose, CA) and Sigma (St Lous. MO ), respectively.
Antibodies to phospho-threonine, ERK, phospho-ERK (Thr202/Tyr204), Akt,
phosphoserine Akt, phospho-p38, and phospho-JNK were from Cell Signaling (Beverly,
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MA) and those for phosphothreonine Akt and EGF receptor were from Upstate (Lake
Placid, NY). NEU3 protein was detected with a monoclonal anti-NEU3 antibody
prepared as previously described (Wang et al., 2002). To assess in vivo Ras activation,
affinity precipitation assays for active (GTP-bound form) Ras were performed using
Raf-1 RBD-agarose (Upstate, NY) according to the manufacturer’s recommendations,
except that the lysis buffer contained 0.25% sodium deoxycholate. The bound Ras-GTP
protein was immunoblotted with anti-Ras antibody. Capture and analysis of images of
protein bands were accomplished using an imaging system equipped with a CCD
camera (VersaDoc 5000, Bio-Rad).
Measurement of Cell Viability and Apoptosis
Cell growth and viability were determined using MTT assays using 96 well culture
plates, and apoptosis was assayed with a TUNEL assay kit (Roche) as recommended by
the supplier. For some experiments, after 24 h of NEU3 siRNA- or NEU3- transfection,
cells were cultured under serum-depleted conditions for 16 h and then apoptosis was
induced by treatment with antiTo evaluate the caspase-3 activity level, cell homogenates were immunoblotted with
antibody for cleaved caspase-3 (Asp-175, Cell Signaling Technology) as above.
Glycolipid analysis by TLC.
Glycolipids were extracted from cells as described elsewhere (Kakugawa et al., 2002),
fractionated by TLC on HPTLC plates (Baker, Phillipsburg, NJ ) in
CHCl3/CH3OH/0.5% CaCl2 (60:40:9, v/v/v) and visualized with orcinol-H2SO4. The
analysis of ceramides was performed in CHCl3/ HOAc (9:1, v/v) and lipids were stained
with 3% cupric acetate in 8% phosphoric acid (Macara et al., 1983). Densitometric
analysis of the band intensities was performed using the NIH Image software.
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