1(-) and a TA cloning kit from Invitrogen (San Diego, CA, USA); E. coli (competent cells) JM109 from Toyobo (Tokyo, Japan); restriction endonucleases, BamHI, EcoRI, and
G418 (geneticin) from Gibco; cell transfection and NucleoBond plasmid kits from GE Healthcare (Piscataway, NJ, USA); AmpliTaq Gold™ and a Bigdye™ terminator cycle sequencing ready reaction kit from Perkin-Elmer/Applied Biosystems (Foster City, CA, USA); DMEM and fetal bovine serum (FBS) from Hyclone (Logan, UT, USA); trypsin, ethylenediamine tetraacetic acid (EDTA), dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) from Amresco (Solon, OH, USA); SABC test kit from Boshide Biotech Co (Wuhan, China); α-L-fucosidase and methylene blue from Sigma (St. Louis, MO); PI3K inhibitor LY294002 from
Promega (Madison, WI); primers and Reverse Transcription Polymerase Chain Reaction (RT-PCR) reagents are products Enzalutamide molecular weight of TaKaRa Biotechnology Co. Ltd (Dalian, China); mouse anti-human Lewis y monoclonal antibody from Abcam (UK); rabbit anti-human IgM monoclonal antibody, PCNA and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Akt and p-Akt from Cell Signaling Technology, MM-102 cost Inc. (Beverly, MA, USA); protein content in cell lysates was measured by the BCA method (Beyotime, China). Cell culture Cells were cultured in DMEM supplemented with 10% FBS at 37°C under 5% CO2 in humidified air. Construction of plasmid and generation of stably Akt inhibitor transfected cell lines The human α1,2-fucosyltransferase gene (FUT-1) was amplified by PCR with human leukocyte genomic DNA as a template and primers according to the human FUT-1 gene sequence (GenBank Accession Number: M35531), sense primer, 5′-CATGTGGCTCCGGAGCCATCGTC-3′, and antisense primer,
5′-GCTCTCAAGGCTTAGCCAATGTCC-3′, under the following conditions: denaturation at 94°C for 9 min, followed by 25 cycles of 94°C, 1 min, 65°C, 1.5 min, and 72°C, 2 min, and then extension at 72°C for 10 min. The PCR products were ligated into the pCR2.1 vector to clone FUT-1 gene, and its DNA sequence was determined by means of the dideoxynucleotide chain-termination method Amobarbital with the BigDye terminator cycle sequenceing ready reaction kit and a DNA sequencer (ABI Genetic Analyzer; Perkin-Elmer/Applied Biosystems). Then the FUT-1 gene in pCR2.1 was cut out by digestion with restriction enzymes, BamHI and EcoRI, and ligated into the BamHI and EcoRI sites of the pcDNA3.1 vector (pcDNA3.1-hFUT). pcDNA3.1-hFUT and the vector alone were transfected into RMG-I cells with a vector transfection kit, according to the instructions for the kit to establish RMG-I-H and RMG-I-pcDNA3.1 cells, respectively. The resultant transfectants were initially selected by cultivation with medium containing an aminoglycoside antibiotic, G418, at 400 μg/ml concentration, and were maintained at 200 μg/ml for 15 days.