Amplification and Sequencing of SLC5A8. The primers used for RT-PCR assay of a SLC5A8 fragment were 5'-TCCGAGGTCTACCGTTTTG and 5'-GGGCAGGGGCATAAATAAC. The PCR parameters were 35 cycles of 95˚C for 45 s, 54˚C for 45 s, 72˚C for 60 s, and 72˚C for 10 min, and 4˚C to cool. The full-length SLC5A8 ORF was amplified by using primers 5'-TCCGGGATAAGAAGTGCG and 5'-TAGTATCAGAGCAGCTTCACAAAC. A GC-rich cDNA polymerase kit (CLONTECH) was used, and PCR parameters were 35 cycles of 95˚C for 45 s, 62˚C for 45 s, 72˚C for 90 s, and 72˚C for 10 min, and 4˚C to cool. Sequencing primers were 5'-TTTGTGGTGGTCATCAGCG, 5'-GGGCAGGGGCATAAATAAC,5'-AGGCTGTGGTGATGCAAGGT, 5'-TTAATGCCTTAGCAGCAG,and 5'-CCTCCACTTCCTGAGAGAAC.

Constructs. To construct the V5-tagged SLC5A8 expression vector, PCR primers 5'-TCCGGGATAAGAAGTGCG and 5'-TCTAGTATCAGAGCAGCTACACAA were used. The PCR conditions were the same as employed for amplification of the full-length ORF. PCR products were cloned into pcDNA3.1/V5-His-TOPO vector (Invitrogen).

Serum DNA Purification. Blood was drawn into red/gray vacutainer collection tubes and allowed to clot for 2 h. It was then spun in a clinical tabletop centrifuge for 15 min at 3,000 rpm at room temperature. Serum was collected by using a sterile pipette, divided into 1-ml aliquots, and stored at minus 80˚C. Serum DNA from patients was purified as described (1).

Western Analysis. Approximately 10⁷ cells were lysed in cell lysis buffer [50 mM TrisˇHCl, pH 7.4/1 mM EGTA/1% Nonidet P-40/0.25% sodium deoxycholate/150 mM NaCl]. Equal amounts of protein were subjected to SDS/PAGE and then transferred to a poly(vinylidene difluoride) nylon membrane (Millipore), which was probed with 1:200 dilution of mouse anti-V5 monoclonal antibody (Invitrogen). Immune complexes were visualized with an ECL Plus Western blotting detection kit (Amersham Biosciences) after incubation with horseradish peroxidase-coupled secondary antibody (Santa Cruz Biotechnology).

Xenopus Oocyte Experiments. The Xenopus oocyte SLC5A8 expression vectors were constructed as follows. Xenopus expressing vector pT7TS (2) was digested with SpeI, while SLC5A8 expressing plasmid on the backbone of pCDNA3.1-V5/His-TOPO was cut by AgeI. The right fragment was purified, and the ends were filled by using Klenow DNA polymerase. The pT7TS plasmid was cut by BglII and SLC5A8 vector with BamHI. To reduce self-ligation, the former were treated with CIP and the fragments were ligated.

Linearized cDNA was used as a template to make capped cRNA. Fifty nanoliters of water (control) or RNA solution (50 nl of 0.6 μg/μl or ≈30 ng of SLC5A8 cRNA) was injected into stage V/VI Xenopus oocytes. Oocytes were studied 3–6 days after injection. Oocyte electrophysiology experiments were carried out as described (3).

Immunofluorescence. Oocyte expression of SLC5A8 was verified by immunolocalization of the V5 tag in oocyte cryosections as described (4). Briefly, oocytes were fixed with PLP-paraformaldehyde, embedded in OCT compound, and cryosectioned. SLC5A8 cRNA-injected Xenopus oocyte sections were incubated with 1:500 Anti-V5 antibody (Invitrogen), followed by a second antibody goat anti-mouse-Cy3 1:500 incubation. Stained sections were visualized by transmission and epifluorescence microscopy. The V5 staining was coincident with the plasma membrane.

Sodium Bisulfite Treatment, Flanking PCR, and Methylation-Specific (MS)-PCR. Sodium bisulfite treatment to convert unmethylated cytosine to thymidine was similar to that described in ref. 1. Primers that flank the SLC5A8 exon 1 CpG island are 5'-CGTGAAGGTAAAGATGTTAAAAATG and 5'-ACAACTAAAAACTCCAATTCTCATC. PCRs were carried out by using a hot start at 95˚C for 7 min and the following cycling parameters: 35 cycles of 95˚C for 45 s, 56˚C for 45 s, 72˚C for 45 s, and 72˚C for 10 min, and 4˚C to cool. Primers to amplify the methylated allele are AS-meth-442-459s (5'- TCGAACGTATTTCGAGGC) and AS-meth-550as (5'-ACAACGAATCGATTTTCCG). PCR parameters were 31 cycles of 95˚C for 45 s, 56˚C for 45 s, 72˚C for 45 s, and 72˚C for 10 min, and 4˚C to cool. Primers to amplify the unmethylated allele are AS-unmeth-442s (5'-TTGAATGTATTTTGAGGTG) and AS-unmeth-542as (5'-TCAATTTTCCAAAATCCC). PCR parameters were 31 cycles of 95˚C for 45 s, 46˚C for 45 s, 72˚C for 45 s, and 72˚C for 10 min, and 4˚C to cool.

Methylation-Specific Real-time PCR. The same MS-PCR primers as above (As-meth-442-459s and As-meth-550as) were first used to amplify a bisulfite converted methylated SLC5A8 exon 1 template. A fluorogenic hybridization probe was designed by using sequences specific for the sodium bisulfite-converted SLC5A8 methylated template. The sequence was 5'-6FAM-CAA CGA CGA ATA CAA AAA CGA CTA CCA AC-BHQ-2-3'. Bisulfite-converted sequences from the MYOD1 gene were used as an internal reference as described (5). Primers and probes for MYOD1 were forward 5'-CCA ACT CCA AAT CCC CTC TCT AT-3', reverse 5'-TGA TTA ATT TAG ATT GGG TTT AGA GAA GGA-3', and probe 5'-6FAM-TCC CTT CCT ATT CCT AAA TCC AAC CTA AAT ACC TCC-BH-2-3'. All the above primers and probes were synthesized by Integrated DNA Technologies (Coralville, IA). For the gene of interest, SLC5A8, the reaction mix contained 600 nM primer, 200 nM probe, 5.5 mM Mg²⁺, and 1× Supermix from Bio-Rad. The total volume was 25 μl. For the MYOD1 gene, the reaction mix contained 400 nM primer, 200 nM probe, 3 mM Mg²⁺, and 1× Supermix from Bio-Rad. The total volume was also 25 μl. Thermal cycling was initiated with 50ΊC for 2 min, then 95ēC for 10 min, followed by 55 cycles of 95ēC for 15 sec and 60ēC for 1 min. PCR was performed in separate wells for each probe/primer set. Each plate contained multiple positive controls, negative controls, and water blanks. Colon cancer cell line RKO was used for a positive control, and V9M was used as a negative control. Serial dilutions of RKO DNA were used to create a standard curve. SLC5A8 methylation was determined as the ratio of SLC5A8:MYOD1 = 2 exp- (CT _SLC5A8 – CT _MYOD1).

Aberrant Crypt Foci (ACF). ACF (6–8) were isolated from grossly normal human colonic mucosa according to the method of Bird et al. (9). Strips of human colonic mucosa, stored over liquid nitrogen, were thawed rapidly in 1% paraformaldehyde and fixed flat in 70% ethanol for 30 min at 4°C (9). The colonic strips were stained for 2 min in 0.2% methylene blue (Chroma-Gesellschaft Schmid & Co, distributed by Roboz Surgical Instrument, Washington, DC) in 0.1 M sodium phosphate buffer (pH 7.4), rinsed in 1% paraformaldehyde for 15 min, transferred mucosal-side-up to a glass slide, and viewed at ×30 magnification under a dissecting microscope. The ACF were teased from the mucosa with microdissection forceps (FWR #55, Dumont Bio Inox Forceps, 0.05 × 0.02 mm tips), placed in microfuge tubes, and stored over liquid nitrogen. The control for each ACF was a similar number of microscopically normal crypts teased from the same mucosa.

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