Fig 2: Expression of three sialyl-Lewis oligosaccharides and relative FUTs and the inhibition effect of monoclonal antibody on cell proliferation. (A) MHCC97 human hepatocellular carcinoma cells and THLE-2 normal liver cells were assayed by flow cytometry using three monoclonal antibodies targeting sialyl-Lewis antigens. PBS, cells treated with PBS and without antibodies as a control; KM93, cells treated with mAb against SLex; FH6, cells treated with mAb against SDLex; CA19-9, cells treated with mAb against SLea. In the flow cytometry histograms, the areas in green show the number of unstained cells and the areas outlined in red represent cells binding to mAb. (B) Quantitative analysis of the expression of sialyl-Lewis oligosaccharides by calculation of mean fluorescence intensity. THLE-2 cells were used as a control. (C) THLE-2 cells and HT29 cells were assayed by flow cytometry using monoclonal antibody against SLea antigen to confirm the high affinity of the SLea antibody. (D) mRNA expression of FUT7 and FUT6 were detected by reverse transcription-quantitative polymerase chain reaction analysis. The relative gene mRNA level was normalized to the respective glyceraldehyde-3-phosphate dehydrogenase level. THLE-2 cells were used asa control. (E) Protein expression levels of FUT7 and FUT6 protein were evaluated by western blot analysis (above) and quantitative analysis of relative protein levels was performed (below). THLE-2 cells were used as a control. **P<0.01 (n=3), vs. THLE-2 Control. (F) Effect of monoclonal antibodies on the proliferation of THLE-2 cells and MHCC97 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide assay showed that MHCC97 cell proliferation was significantly suppressed by KM93, a mAb against SLex. FUT, fucosyltransferase; SLex, sialyl-Lewis X, SLea, sialyl-Lewis a; SDLe X, dimeric SLe; mAb, monoclonal antibody.

Fig 3: Changes in the expression of FUT7 and SLex of MHCC97 cells following transfection with FUT7 siRNAs. siRNA targeting FUT7 was transfected into MHCC97 cells and the interference rate was confirmed by RT-qPCR and western blot analyses. Flow-cytometric analysis was used to detect the expression of SLex. (A) mRNA level of FUT7 detected by RT-qPCR analysis following FUT7-siRNA transfection. (B) Protein level of FUT7 detected by western blot analysis following FUT7-siRNA transfection (above) and quantitative analysis of the protein level of FUT7 following FUT7-siRNA transfection below). (C) Flow cytometric analysis of cell surface expression of SLex following FUT7-siRNA transfection. (D) Quantitative analysis of the expression of SLex by calculating the mean fluorescence intensity. *P<0.05 (n=3), vs. untransfected control cells; **P<0.01 (n=3), vs. untransfected control cells. FUT, fucosyltransferase; SLex, sialyl-Lewis X; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; siRNA, small interfering RNA; siNC, scramble-siRNA transfected cells; Control/Ctrl, untransfected cells.

Fig 4: Knocking down FUT7 affects cell survival via various intracellular signaling mediators. MHCC97 cells were transfected with FUT7 siRNAs. After 1 h, inhibitors of signaling molecules were added to the culture medium, and the cells were cultured for a further 48 h for RNA analysis and 72 h for protein analysis. (A) mRNA levels of FUT7 were quantified by reverse transcription-quantitative polymerase chain reaction following treatment with inhibitors. (B) Following treatment with the inhibitors, cell survival was quantified using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide assay in the THLE-2 normal liver cells and MHCC97 hepatocarcinoma cells. (C) Cells were permeabilized and centrifuged to isolate the cytosolic proteins and particulate-conjugated proteins. Results of western blot analysis of the target proteins are shown with profiles. (D) Quantitative analysis of relative protein levels. *P<0.05 (n=3), vs. Control; **P<0.01 (n=3), vs. Control. FUT, fucosyltransferase; Ctrl, control (no inhibitor); ErkI, extracellular signal-regulated kinase inhibitor; siRNA, small interfering RNA; siNC, scramble-siRNA transfected cells; PLCγ, phospholipase Cγ; PLCγ1-C, PLCγ1 in cytoplasm; PLCγ1-M, PLCγ1 conjugated with plasma membrane; p-PLCγ1, phosphorylated PLCγ1 conjugated with plasma membrane; Control, untransfected cells.

Fig 5: SLeX+ CD4+ T Cells Are Enriched for Pathways Involved in T Cell Extravasation and HIV Transcription(A) Relative expression of GCNT1 and FUT7 genes in SLeX-Hi and SLeX-Low CD4+ T cells isolated from four HIV+ ART-suppressed individuals. Mean and SEM are presented, and p values were calculated using paired t tests. FDR was calculated using the Benjamin-Hochberg procedure.(B and C) List of significantly enriched pathways (B) and functions (C) found by IPA among 1,086 genes significantly different (FDR < 5%, at least 2-fold) between SLeX-Hi and SLeX-Low CD4+ T cells. N, number of genes significantly changed in the pathway; % up, percentage of genes expressed at higher levels in SLeX-Hi compared with SLeX-Low CD4+ T cells; Z, activation Z score of the pathway or function predicted by IPA based on the direction of changes and contribution of membership genes; Positive, activated; negative, inhibited in SLeX-Hi.(D and E) Genes differentially expressed (FDR < 5%) in SLeX-Hi compared with SLeX-Low CD4+ T cells that belong to (D) leukocyte extravasation signaling or (E) NF-?B signaling. Red dots are genes expressed at higher levels in SLeX-Hi compared with SLeX-Low CD4+ T cells, whereas blue dots are genes expressed at lower levels in SLeX-Hi compared with SLeX-Low CD4+ T cells.