･Supplemental figure regend
Representativeimmunohistochemistry of ST6GalNAc I in gastric tissues.
Representative photomicrographs of anti-ST6GalNAc I antibody-stained gastirc specimens obtained from gastric carcinoma of the diffuse type (A) and gastric carcinoma of the intestinal type (B). Pictures taken at original magnification (x200, x400) and magnified images of the corresponding areas in boxes are presented below as indicated. Bar represent 100 μm.This examination follows the tenets and regulations of the Declaration of Helsinki and has been approved by the Institutional Review Board at SapporoMedical University.
Overexpression of ST6GalNAc Iby ST6GalNAc I plasmid promote cell proliferation, cell invasion and migration in MKN74 cells.
A,The cell proliferation of untreated, random siRNA- or ST6GalNAc Iplasmid-transfected MKN74 cells was measured by the WST-1 assay. The results were from triplicate measurements from two independent experiments. *P<0.05. B,In vitro migration assays were done using a modified Boyden chamber system used MKN74 cells. Equal numbers (3 x105) of transfected cells were placed in the top chamber and allowed to migrate for 24 hours. Migrated cells were stained with methylene blue and counted.Representative photographs are shown at leftmagnification. The right panel shows the number of migrated cells that were transfected as indicated. The results were from triplicate measurements from two independent experiments. *P<0.05. C,Invasion assays of ST6GalNAc Iplasmid-transfected MKN74 cells. After 48 hours, invading cells were stained with methylene blue and counted . The right panel shows the number of invaded cells that were transfected as indicated.*P<0.05. Bar represent 100 μm.
Expression levels of mRNA associated with tumor metastasis in MKN45 cells treated with ST6GalNAc I siRNA.
A, Heat map represents the fold increase or decrease in mRNA expression of 89 genes listed in columns (A-G) and in rows (1-12). The expression levels of these genes in ST6GalNAc I siRNA-treated MKN45 cells were closely associated with tumor metastasis when compared with the gene levels in random siRNA-treated MKN45 cells. Color-coded graphs were generated by using the Web-based program of the RT2 profiler PCR Array Data Analysis. The green color indicates low expression and the red color indicates high expression. B, list of genes showing 1.5-fold or greater fold difference were summarized. ΔΔCt values: positive values represent enhanced expression; negative values represent suppressed expression. A 1.5-fold change in mRNA expression was considered as significant when compared with the random siRNA-transfected control.
Overexpression of ST6GalNAc Iby ST6GalNAc I plasmid in gastric cancer cells.
A,MKN45 cells were transfected with an ST6GalNAc I-expressing vector and expression of ST6GalNAc I was evaluated by qPCR. ST6GalNAc I expression levels were calculated as ratios in relation to expression in the mock transfectant. B, Immunoblot analysis of ST6GalNAc I expression levels in MKN45 cells transfected with mock vector or ST6GalNAc I expression plasmid. β-Actin was used as a loading control.