Journal: Experimental and Therapeutic Medicine

Article Title: Silencing GnT-V reduces oxaliplatin chemosensitivity in human colorectal cancer cells through N-glycan alteration of organic cation transporter member 2

doi: 10.3892/etm.2020.9560

Figure Lengend Snippet: GnT-V expression is associated with oxaliplatin chemosensitivity in CRC cells. (A) The mRNA expression of MGAT5 was examined by reverse transcription-quantitative PCR. (B) Protein levels of GnT-V in CRC cell lines were measured by western blotting. (C) Cells were treated with the indicated concentrations of oxaliplatin for 48 h, and oxaliplatin sensitivity was determined based on cell death using the trypan blue method. * P<0.05 vs. HT29 group and # P<0.05 vs. HCT116 group. (D) Acute treatment with 1.5 µM oxaliplatin for 24 or 48 h did not lead to changes in GnT-V expression levels in CW-2 and HT29 cells. (E) CW-2 and HT29 cell lines were exposed to long-term oxaliplatin treatment to obtain stably resistant lines, named CW-2/R and HT29/R, respectively. Endogenous MGAT5 expression was increased in the stably resistant cell lines as compared with the parental cells at the mRNA level. (F) GnT-V expression and β-1,6-oligosaccharide branches were detected by western blot and lectin blot analyses, respectively, in wild-type and oxaliplatin-resistant cell lines. The graphs depict results from three independent experiments each performed in triplicate. Results are presented as the mean ± SEM. * P<0.05, # P<0.05 and ** P<0.01. GnT-V, N-acetylglucosaminyltransferase V; CRC, colorectal cancer; MGAT5, the gene encoding GnT-V; OXA, oxaliplatin; UT, untreated; PHA-L, phytohemagglutin-L.

Article Snippet: For lectin blot assay, blocked membranes were incubated with biotinylated phytohemagglutin-L (PHA-L) lectin (dilution 1:400; Vector Laboratories, Inc.) for 1 h at room temperature.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Stable Transfection

Journal: Experimental and Therapeutic Medicine

Article Title: Silencing GnT-V reduces oxaliplatin chemosensitivity in human colorectal cancer cells through N-glycan alteration of organic cation transporter member 2

doi: 10.3892/etm.2020.9560

Figure Lengend Snippet: Cells with GnT-V knockdown exhibit enhanced survival and cell viability upon exposure to oxaliplatin. (A) and (B) shRNA mediated GnT-V knockdown and β-1,6-oligosaccharide reduction in (A) CW-2 and (B) CW-2/R cells as depicted by western blotting and lectin blotting, respectively, compared with the respective parental cell lines and NC cells. (C) Cells were exposed to indicated concentrations of oxaliplatin (0.25-16 µg/ml) for 48 h, and cell viabilities were determined by Cell Counting Kit-8 assay. Representative images of (D) wild-type and (E) drug-resistant cells showing that oxaliplatin-treated GnT-V knockdown cells had reduced chemosensitivity, resulting in an increased colony-forming potential compared with NC cells. Results are presented as the means ± SEM from three independent experiments. * P<0.05. GnT-V, N-acetylglucosaminyltransferase V; shRNA, short hairpin RNA; shRNA#1 and #2, shRNAs for knockdown of GnT-V; NC, negative control; OXA, oxaliplatin; PHA-L, phytohemagglutin-L; UT, untreated.

Article Snippet: For lectin blot assay, blocked membranes were incubated with biotinylated phytohemagglutin-L (PHA-L) lectin (dilution 1:400; Vector Laboratories, Inc.) for 1 h at room temperature.

Techniques: shRNA, Western Blot, Cell Counting, Negative Control

Journal: Experimental and Therapeutic Medicine

Article Title: Silencing GnT-V reduces oxaliplatin chemosensitivity in human colorectal cancer cells through N-glycan alteration of organic cation transporter member 2

doi: 10.3892/etm.2020.9560

Figure Lengend Snippet: OCT2 acts as a substrate of GnT-V and affects the cytotoxic response to oxaliplatin in CRC cells. (A and B) GnT-V knockdown did not lead to marked changes in OCT2 expression in CW-2 and CW-2/R cells as compared with the respective wild-type and NC cells. (C) Reduced cytotoxic responses to oxaliplatin were observed after treatment with 100 µM cimetidine. (D) Lectin precipitation was performed with PHA-L-bound agarose, followed by western blotting with an anti-OCT2 antibody. Data were obtained from triplicate experiments and are presented as the mean ± SEM. * P<0.05. OCT2, organic cation transporter member 2; GnT-V, N-acetylglucosaminyltransferase V; NC, negative control; shRNA#2, short hairpin RNA for knockdown of GnT-V; OXA, oxaliplatin; IP, lectin precipitate; IB, immunoblot; PHA-L, phytohemagglutin-L.

Article Snippet: For lectin blot assay, blocked membranes were incubated with biotinylated phytohemagglutin-L (PHA-L) lectin (dilution 1:400; Vector Laboratories, Inc.) for 1 h at room temperature.

Techniques: Expressing, Western Blot, Negative Control, shRNA

Journal: PLoS ONE

Article Title: Epithelial-to-Mesenchymal Transition of RPE Cells In Vitro Confers Increased β1,6- N -Glycosylation and Increased Susceptibility to Galectin-3 Binding

doi: 10.1371/journal.pone.0146887

Figure Lengend Snippet: (A-C) Cultured human RPE cells were treated with swainsonine to block N-glycan elongation followed by incubation with Gal-3 or plant lectins for 30 minutes at 4°C as described in the Materials and Methods Section. (A) Effectiveness of swainsonine treatment is shown by decreased staining with the plant lectin PHA-L ( dotted line ) compared with control treated cells ( thick line ). (B) Reduction of complex-type N-glycans on RPE cells results in decreased Gal-3 binding ( dotted line ). (C) Binding of the control lectin WGA is not modified. (D-F) Treatment of cultured RPE cells with deoxymannojirimycin (DMNJ), another inhibitor of N-glycan branching, reduces binding of the plant lectin PHA-L ( dotted line ) (D) and Gal-3 ( dotted line ) (E) compared with control treated cells ( thick line ) whereas binding of the control lectin WGA ( dotted line ) is not affected (F). (G-I) RPE cells were treated with BenzylGalNAc, which inhibits elongation of O-glycans and can compete with sialyltransferases resulting in decreased O-glycan sialylation, allowing increased branching of O-glycans. Decreased O-glycan sialylation is shown by increased staining with the plant lectin PNA ( dotted line ) compared with untreated controls ( thick line ) (G). (H) Increased accessibility of branched O-glycans does not alter binding of Gal-3 to RPE cells ( dotted line ), showing that branched O-glycans are not required for Gal-3 binding. (I) Binding of PHA-E as a control lectin is not altered by benzylGalNAc treatment. (J-L) Treatment of RPE cells with neuraminidase increases Gal-3 binding to the surface of cultured human RPE cells. Cultured, myofibroblastic RPE cells were incubated with or without Vibrio Cholera neuraminidase for 30 minutes at 37°C. (J) Binding of MAL-2 (specific for α2,3 sialic acid residues) ( thick line ) and (L) SNA (specific for α2,6 sialic acid residues) ( thick line ) confirms the presence of sialic acid residues on glycans on the RPE cell surface. Both, MAL-2 and SNA binding, is reduced after treatment with neuraminidase ( dotted line ). Binding of Gal-3 (K) to cultured human RPE is increased by removal of sialic acids ( dotted line ). Results are representative for three independent experiments.

Article Snippet: Blots were probed with biotin-conjugated Phaseolus vulgaris leukoagglutinin (PHA-L), or Phaseolus vulgaris erythroagglutinin (PHA-E), or Concavalin A (ConA), Wheat germ agglutinin (WGA), or Ricinus communis agglutinin (RCA), or Sambuccus nigra lectin (SNA), or Griffonia simplificolia lectin-1 (GSL-1), or Peanut agglutinin (PNA), or Maackia amurensis lectin (MAL-2), or Lycopersicum esculentum lectin (LEL), or Jacalin (JAC), respectively (all from Vector Labs), diluted 1:1,000 in PBS 0.05% Tween 20 overnight at 4°C.

Techniques: Cell Culture, Blocking Assay, Incubation, Staining, Binding Assay, Modification

Journal: PLoS ONE

Article Title: Epithelial-to-Mesenchymal Transition of RPE Cells In Vitro Confers Increased β1,6- N -Glycosylation and Increased Susceptibility to Galectin-3 Binding

doi: 10.1371/journal.pone.0146887

Figure Lengend Snippet: (A) Flow cytometric analysis of Gal-3 binding to native and myofibroblastic RPE cells. Binding of Gal-3 ( gray ) to native RPE cells was only slightly above background, whereas Gal-3 binding to myofibroblastic RPE cells was evident. ConA binding was markedly above background in both cell populations. Histograms represent the number of cells versus relative fluorescence intensity. Experiments have been repeated two times. (B) Lectin histochemistry of human RPE cells in situ. Human cadaver eyes were incubated with biotinylated lectins as indicated on the left and binding was visualized by incubation with streptavidin-coupled peroxidase and Vector VIP substrate™ (purple color). In control sections with the VIP substrate alone the RPE can be easily discerned by the characteristic brownish pigment (third panel from the top). RPE cells and extracellular matrix reacted strongly with ConA (top panel ), whereas PHA-L ( second panel from the top) did not recognize native RPE cells and exhibited a staining pattern comparable to that of substrate alone. Biotinylated Gal-3 (purple color, fourth panel ) did not bind to native RPE in situ and staining patterns resembled closely the situation in untreated negative control eyes. RPE, retinal pigment epithelium; BM, Bruch´s membrane; CH, choriocapillaris.

Article Snippet: Blots were probed with biotin-conjugated Phaseolus vulgaris leukoagglutinin (PHA-L), or Phaseolus vulgaris erythroagglutinin (PHA-E), or Concavalin A (ConA), Wheat germ agglutinin (WGA), or Ricinus communis agglutinin (RCA), or Sambuccus nigra lectin (SNA), or Griffonia simplificolia lectin-1 (GSL-1), or Peanut agglutinin (PNA), or Maackia amurensis lectin (MAL-2), or Lycopersicum esculentum lectin (LEL), or Jacalin (JAC), respectively (all from Vector Labs), diluted 1:1,000 in PBS 0.05% Tween 20 overnight at 4°C.

Techniques: Binding Assay, Fluorescence, In Situ, Incubation, Plasmid Preparation, Staining, Negative Control

Journal: Journal of Biomedicine and Biotechnology

Article Title: Explanting Is an Ex Vivo Model of Renal Epithelial-Mesenchymal Transition

doi: 10.1155/2011/212819

Figure Lengend Snippet: Lectin staining characteristics of cell outgrowths, 10 days after explanting. The majority of cells stain for Pha-E and Pha-L, lectins specific for the proximal tubular epithelium. (a) Arachis hypogaea (AH); (b) Bandeiraea simplicifolia I (BSL-I); (c) Phaseolus vulgaris erythroagglutinin (Pha-E); (d) Phaseolus vulgaris leukoagglutinin (Pha-L). Scale bar = 25 μ m.

Article Snippet: Tissue sections were incubated for 2 hr with biotin-conjugated phaseolus vulgaris leukoagglutinin (Pha-L; Vector) (proximal tubules and thick loop of Henle), phaseolus vulgaris erythroagglutinin (Pha-E; Vector) (proximal tubules), Bandeiraea simplicifolia I (BSL-I; Sigma) (collecting ducts, vasa recta ), or Arachis hypogaea (Sigma) (distal convoluted tubules and collecting ducts).

Techniques: Staining

Journal: The Journal of Biological Chemistry

Article Title: MicroRNA-424 Predicts a Role for β-1,4 Branched Glycosylation in Cell Cycle Progression *

doi: 10.1074/jbc.M115.672220

Figure Lengend Snippet: Loss of MGAT4A activity does not affect GlcNAc-β-1,6 branching. A, biosynthetic pathway of tetraantennary branched N-glycans. Binding sites of PHA-L are indicated. B, analysis of PHA-L binding to the Consortium of Functional Glycomics (CFG) glycan array v. 5.0 indicates a preference for GlcNAc-β-1,6 branched products. The graph shows the data for 100 μg/ml biotinylated-PHA-L (Vector Laboratories) binding to glycans bearing only β-1,4 branching (β-(1,4)) and the corresponding β-1,6 glycans (β-(1,6)). Data are given as fluorescence intensity. C, knockdown of MGAT4A in MCF-7 cells shows no significant change in MGAT4B or MGAT5 mRNA levels by qRT-PCR. The graph shows -fold change mRNA expression compared with NTC. n = 3 biological replicates. Error bars represent the S.D. D, fluorescence microscopy of PHA-L stained MGAT4A-KD and NTC cells. No significant loss of PHA-L binding were observed. Images shown are representative of two biological replicates, five images per replicate.

Article Snippet: The graph shows the data for 100 μg/ml biotinylated-PHA-L (Vector Laboratories) binding to glycans bearing only β-1,4 branching (β -(1,4) ) and the corresponding β-1,6 glycans (β -(1,6) ).

Techniques: Activity Assay, Binding Assay, Functional Assay, Plasmid Preparation, Fluorescence, Quantitative RT-PCR, Expressing, Microscopy, Staining