1D and E). cells. The expression of SLea was only detected in trace quantities. Fucosyltransferase (FUT) is the key enzyme of the fucosylation step in the biosynthesis of sialyl-Lewis oligosaccharide antigens. Therefore, the present study investigated the expression of FUTs. It was found that the mRNA and protein expression levels of FUT7 were high in the MHCC97 HCC cell line compared with levels in normal liver cells. FUT6 was also expressed at a high level, although the difference was not statistically significant between MHCC97 cells and normal liver cells. No expression of FUT3 was detected. The results were consistent with the change insialyl-Lewis antigens. The effects of FUT7 small interfering (si)RNA transfection on the expression of FUT7, expression of SLex and MHCC97 cell proliferation were also examined. Following FUT7 siRNA transfection, the expression of FUT7 was markedly downregulated, as determined by western blot and reverse transcription-quantitative polymerase chain reaction methods. The results from flow cytometry showed that the synthesis of SLex was also inhibited, which was consistent with the downregulated expression of FUT7. MHCC97 cell proliferation was also significantly inhibited following FUT7 siRNA transfection, which was correlated with suppression of the S-phase in cell cycle progression. By using inhibitors of various signaling pathways, it was found that the knockdown of FUT7 inhibited the activation of phospholipase C (PLC) by inhibiting the translocation and phosphorylation of PLC. In conclusion, the results suggested that FUT7 has animportant functional role in human HCC cell proliferation by controlling cell cycle progression via the PLC/extracellular BIBF0775 signal-regulated kinase signaling pathway. The inhibition of SLex and FUT7 siRNA transfection may provide a novel therapeutic methodology to treat tumors that express SLex glycoconjugates. forward 5-CAT TTC TGC TGC CTC AGG-3 and reverse 5-GGG CAA GTC AGG CAA CTC-3; human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward 5-GAA GGT GAA GGT CGG AGT C-3 and reverse 5-GAA GAT GGT GAT GGG ATT TC-3. The relative mRNA expression levels of FUT7 and FUT6 were normalized to the endogenous mRNA expression of GAPDH. Western blot analysis RIPA buffer (Sigma; EMD Millipore) with protease inhibitor (Sigma; EMD Millipore) was used to lyse cells (26). The BIBF0775 protein was collected, and a BCA Pierce Assay (Thermo Fisher Scientific, Inc.) was used for the quantification of protein concentration. Subsequently, 50 em /em g of protein from each sample was denatured and resolved on a 10% SDS-PAGE gradient gel (EMD Millipore), and then electro-blotted onto a PVDF nitrocellulose membrane (EMD BIBF0775 Millipore). The PVDF membranes were then incubated with 5% non-fat milk for 1 h at room temperature, and then incubated with anti-FUT7 (1:1,000, cat. no. MAB64091), anti-PLC1 (1:500, cat. no. MAB8137) and anti-phosphorylated PLC1 (1:500, cat. no. MAB74541) which were purchased from Bio-Techne China (Shanghai, China), anti-FUT6 (1:1,000, cat. no. NBP1-57936; Novus Biologicals, LLC, Littleton, CO, USA), or anti–actin antibody (1:1,000, cat. no. MAB8929; Bio-Techne China) at 4C overnight. Following washing with TBST, the membrane was incubated with HRP-conjugated secondary antibodies (1:3,000, cat. no. HAF008; Bio-Techne China) for 1.5 h at room temperature. Finally, the signals were developed by enhanced chemiluminescence (Pierce, Thermo Fisher Scientific, Inc.). Images of the results were captured and the images were scanned. The optical density of each protein band was quantified by a scanning densitometer and Quantity One software, version 4.4.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each lane of protein band density was normalized with corresponding -actin density. The cytosolic protein was isolated from particulate- conjugated protein using a digitonin separation method (29). The cells were collected and resuspended in 1 ml saline solution (1 mM EDTA, 150 mMNaCl, 1 mM PMSF, 2 mM EGTA, 1 em /em g/ml aprotinin, 10 em /em g/ml leupeptin, and 100 em /em g/ml digitonin) with occasional agitation for 10 min. The cells were then centrifuged at 13,000 g for 5 min at 4C and the resulting supernatant contained the cytosolic proteins. The cell pellet was dissolved in 1 ml lysis buffer (pH 7.4, 1 mM EDTA, 10 mM NEK3 PBS, 1% Triton X-100, 2 mM EGTA, 1 mM PMSF, 0.1% SDS, 1 em /em g/ml aprotinin, and 10 em /em g/ml leupeptin) and contained the membrane protein (particulate-conjugated proteins). Subsequently, 80 em /em g of protein was separated by SDS-PAGE and transferred onto a PVDF membrane. The expression levels of PLC1 and phosphorylated PLC1 were detected by western blot analysis, as described above. Knockdown of FUT7 in MHCC97 cells by RNAi The expression of FUT7 in MHCC97 cells was silenced using specific siRNAs (Silencer siRNA transfection, Ambion, Thermo Fisher Scientific, Inc.). Scramble.