Fig 1: ST3GAL6 reversed the increase of UBC cell invasion driven by GATA3 depletion. A-B, mRNA and protein expression levels of GATA3 and ST3GAL6 in GATA3 siRNA alone or combined with ST3GAL6 siRNA treated SW780 cells, detected by qRT-PCR (A) and Western blotting (B), respectively. C-D, Global sialylation levels in SW780 cells transfected with GATA3 siRNA (siGATA3) alone or combined with ST3GAL6 siRNA (siST3GAL6), detected by MAL II blotting (C, right panel) and immunofluorescence staining (D, upper panel), respectively. The silver staining blot (C, left panel) was used as a loading control. Hoechst 33253 was used for the nuclei staining (D, middle panel). Scale bars, 50 µm. E, Transwell assay showed the cell invasion capacities of SW780 cells transfected with GATA3 siRNA alone or combined with ST3GAL6 siRNA. Scale bars, 50 µm. F, The working model of GATA3/ST3GAL6 axis in different subtypes of UBC, which is associated with glycogene expression and patients' outcomes. Data were presented as mean ± SD of three independent experiments; ***, p < 0.001.
Fig 2: Biological effects of ST3GAL6 depletion in UBC cells. A-B, mRNA and protein expression levels of ST3GAL6 gene in 5637 and J82 cells transfected with siRNAs targeting ST3GAL6 (siST3GAL6-1 or -2) or control siRNA (siNC), detected by qRT-PCR (A) and Western blotting (B), respectively. C, The cell proliferation of 5637 and J82 cells with the depletion of ST3GAL6 by CCK-8 assay. D-E, Global sialylation levels in 5637 and J82 cells transfected with ST3GAL6 siRNAs, detected by MAL II blotting (D) and immunofluorescence staining (E, upper panel), respectively. The silver staining blots (D) were used as loading controls. Hoechst 33253 was used for the nuclei staining (E, middle panel). Scale bars, 10 µm. F and H, Cell invasion and migration capacities in 5637 and J82 cells transfected with ST3GAL6 siRNAs, detected by transwell invasion (F) and wound healing (H) assays, respectively. Scale bars, 50 µm. G and I, Quantifications of images for transwell invasion (F) and wound healing (H) assays. Data were presented as mean ± SD of three independent experiments; ***, p < 0.001; ns, p = 0.05.
Fig 3: The correlation of GATA3 and ST3GAL6 expression in UBC samples and cells. A-C, The mRNA levels of ST3GAL6 (A) and GATA3 (B) in four subtypes, along with their Pearson correlation (C) in UBC patients from TCGA provisional dataset. D-F, The mRNA levels of ST3GAL6 (D) and GATA3 (E) in four subtypes, along with their Pearson correlation (F) in UBC patients from GSE87304 dataset. G, The protein levels of ST3GAL6 and GATA3 in 9 UBC cell lines by Western blotting. H-I, mRNA and protein expression levels of ST3GAL6 and GATA3 in SW780 and RT4 cells transfected with siRNAs targeting GATA3 (siGATA3-1 or -2) or control siRNA (siNC), detected by qRT-PCR (H) and Western blotting (I), respectively. J, Sequences for ST3GAL6 luciferase reporters, ranging between 13,614 and 14,255 bp from the transcriptional start site of ST3GAL6 (NM_001323360). Wildtype (WT, in blue) and mutant (in orange) GATA binding sites were indicated. K, The activities of ST3GAL6 luciferase reporters (WT and mutant) in the presence of GATA3 expression plasmids (0.2 and 0.4 µg/well) from 5637 cells in 24-well-plate, normalized by activities of co-transfected pRL-CMV. Data were presented as mean ± SD of three independent experiments; ***, p < 0.001; *, p < 0.05.
Fig 4: The association of ST3GAL6 expression and clinical features. A-F, The correlations between ST3GAL6 mRNA levels and tumor stage of UBC patients from GES13507 (A), GES31684 (B), GES32584 (C), GES32894 (D), GES48075 (E), and TCGA provisional (F) datasets. G-J, Kaplan-Meier plot of overall survival of UBC patients in TCGA provisional (G), GES13507 (H), GSE31684 (I) and GSE48075 (J) datasets, stratified by ST3GAL6 expression. K-M, Kaplan-Meier plot of disease-free survival or cancer-specific survival of UBC patients in TCGA provisional (K), GES13507 (L), and GSE32894 (I) dataset, stratified by ST3GAL6 expression. N, Kaplan-Meier plot of cumulative overall survival of UBC patients in our IHC cohort, using the mean value of ST3GAL6 IHC staining scores as the cutoff point. O, IHC staining of ST3GAL6 in UBC patients. The representative images for different staining intensities were shown. ***, p < 0.001; **, p < 0.01.
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