Fig 1: C1GALT1 is a direct target of miR-152 in GC. a Venn diagrams depicting the number of potential miRNAs targeting the 3'UTR of C1GALT1. b Correlation of miR-152 and C1GALT1 expression in GC tissues (Spearman’s rank correlation test). c, d qRT-PCR and Western blot analysis of C1GALT1 expression after miR-152 overexpression or inhibition. e–g Cell proliferation, migration, and invasion were assessed by CCK-8, Transwell migration, and Matrigel invasion assays. h Diagram of the potential binding sequences of miR-152 on the 3'-UTR of C1GALT1 and the mutated sequences of C1GALT1 3'-UTR. i Relative luciferase activity was examined by the dual-luciferase reporter assay. OV: cells transfected with C1GALT1 plasmid. *p < 0.05, **p < 0.01, #p > 0.05 compared with the mimics NC or inhibitor NC group (Student’s t-test or one-way ANOVA)
Fig 2: C1GALT1 regulates the O-glycosylation of integrin a5 in GC. a The number of PNA-binding proteins identified by mass spectrometry was displayed using Venny. b The cell-surface proteins that interacted with PNA were detected using a lectin-based pull-down assay. Membrane proteins were divided into two parts, one for GADPH detection, and the other for PNA-binding measurement. GAPDH was an internal control. c The proteins from whole-cell lysates enriched by PNA were analyzed by immunoblotting with an anti-integrin a5 antibody. d The indicated protein expression was evaluated by Western blot after transfection. e Representative images of integrin a5, p-PI3K, and p-AKT staining in GC samples. Scale bars, 100 µm. f Correlation analysis (Spearman’s rank correlation test). shNC: cells transfected with negative control lentivirus; shRNA: cells transfected with C1GALT1 shRNA2 lentivirus; Mock: cells transfected with empty plasmid; OV: cells transfected with C1GALT1 plasmid; siNC: cells transfected with negative control siRNA; siRNA: cells transfected with integrin a5 siRNA; IB: immunoblot; PD: pull-down
Fig 3: C1GALT1 is transcriptionally activated by SP1 in GC. a Scatter diagrams for C1GALT1 expression versus SP1 expression in GC samples based on the TCGA and GTEx databases(Spearman’s rank correlation test). b Representative images and quantitative analysis of SP1 expression in GC tissues (Spearman’s rank correlation test). Scale bars, 100 µm. c Western blot analysis of SP1 expression in different GC cells. d The effect of SP1 on C1GALT1 expression was detected by Western blot. e Schematic presentation of SP1 binding sites within the promoter region of C1GALT1. f The binding of SP1 to the C1GALT1 promoter was analyzed by ChIP-PCR. g, h Relative luciferase activity was examined by the dual-luciferase reporter assay. i–k Cell proliferation, migration, and invasion were assessed by CCK-8, Transwell migration, and Matrigel invasion assays. siNC: cells transfected with negative control siRNA; si1: cells transfected with SP1 siRNA1; si2, cells transfected with SP1 siRNA2; Ctrl, cells transfected with control plasmid; SP1, cells transfected with SP1 plasmid; OV: cells transfected with C1GALT1 plasmid; WT: Wild-type; MUT: mutant. *p < 0.05, **p < 0.01 compared with the siNC or Ctrl group (Student’s t-test or one-way ANOVA)
Fig 4: C1GALT1 promotes GC cell proliferation, migration, and invasion. a Bioinformatics analysis of C1GALT1 mRNA expression in 38 GC cell lines using the CCLE database. b qRT-PCR analysis of C1GALT1 expression in one normal gastric cell line and six GC cell lines. c Western blot analysis of C1GALT1 expression in different cell lines. d C1GALT1 protein expression was detected by Western blot after transfection. e C1GALT1 mRNA expression was analyzed by qRT-PCR after transfection. f CCK-8 assay was used for proliferation analysis. g Transwell chambers without Matrigel were used for migration analysis. h Matrigel-coated Transwell chambers were used for invasion analysis. shNC: cells transfected with negative control lentivirus; shRNAs: cells transfected with C1GALT1 shRNA lentivirus; Mock: cells transfected with empty plasmid; OV: cells transfected with C1GALT1 plasmid. *p < 0.05, **p < 0.01 compared with the shNC or Mock group (Student’s t-test or one-way ANOVA)
Fig 5: Effects of C1GALT1 on GC proliferation, migration, and invasion are mediated by integrin a5. a–c Cell proliferation, migration, and invasion were assessed by CCK-8, Transwell migration, and Matrigel invasion assays. d Photographs of xenograft tumors. e The tumor growth curve and tumor weight in the nude mice. f Metastatic nodules were photographed and counted. n = 6 mice per group. Mock: cells transfected with empty plasmid; OV: cells transfected with C1GALT1 plasmid; siNC: cells transfected with negative control siRNA; siRNA: cells transfected with integrin a5 siRNA. *p < 0.05, **p < 0.01 compared with the siNC+Mock group (Student’s t-test or one-way ANOVA)
Supplier Page from Abcam for Anti-C1GALT1 antibody