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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) 1 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, 2 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, 3 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. 4 5