Journal: Cell Proliferation

Article Title: Haematopoietic lineage‐committed bone marrow cells, but not cloned cultured mesenchymal stem cells, contribute to regeneration of renal tubular epithelium after HgCl 2 ‐induced acute tubular injury

doi: 10.1111/j.1365-2184.2008.00545.x

Figure Lengend Snippet: Examples of engraftment of haematopoietic lineage marrow cells (HLMCs) and mesenchymal stem cells at 3 days after HgCl2. (a) GFP‐positive control shows proximal tubular cells (PHA‐L, blue colour) co‐stained for GFP (brown colour). (b) GFP‐negative control (wild type) showing no detection of GFP. (c) BMT mouse treated with HgCl2 demonstrating GFP‐positive proximal tubular epithelial cells. Dashed black arrows indicate PHA‐L stained donor HLMC‐derived tubular cells. (d) Male positive control; Y chromosomes were detected by indirect FISH, black arrows point to Y chromosomes (brown dot) in proximal tubular cells histochemically stained with PHA‐L (red colour). (e) Female control showing the lack of Y chromosome detection within proximal tubular cells histochemically stained with PHA‐L. (f) Scattered Y chromosomes (brown dot) in renal interstitial area, not in proximal tubular cells. (g) High magnification of yellow box in (f); a–f, ×400; g, ×600. BMT, bone marrow transplantation; FISH, fluorescence in situ hybridization; PHA‐L, Phaseolus vulgaris leucoagglutinin.

Article Snippet: After GFP staining, tissue sections were microwaved in 10 m m tri‐sodium citrate (pH 6.0) for 10 min and biotin was blocked using a biotin blocking kit (DAKO), and then incubated for 45 min with biotinylated Phaseolus vulgaris leucoagglutinin (PHA‐L) 1/100 (B‐1115, Vector Laboratories, Burlingame, CA, USA) to detect proximal convoluted tubules, or biotinylated peanut agglutinin (PNA) 1/800 (B‐1075, Vector Laboratories) to detect distal convoluted tubules.

Techniques: Positive Control, Staining, Negative Control, Derivative Assay, Transplantation Assay, Fluorescence, In Situ Hybridization

Journal: Cell Proliferation

Article Title: Haematopoietic lineage‐committed bone marrow cells, but not cloned cultured mesenchymal stem cells, contribute to regeneration of renal tubular epithelium after HgCl 2 ‐induced acute tubular injury

doi: 10.1111/j.1365-2184.2008.00545.x

Figure Lengend Snippet: (a) Changes in the abundance of GFP‐positive cells within the PHA‐L stained cell population in control mice and mice treated with HgCl2 (n = 5 per treatment time point). *P < 0.05 versus the same group at day 0; +P < 0.05 versus group CON at the corresponding time point. The percentages of GFP‐positive cells were adjusted based on a correction factor derived from the actual cell count of 41% of PHA‐L stained cells being GFP‐positive in GFP donor mice. (b) Changes in the 3H‐LI of PHA‐L stained proximal tubular cells of: combined indigenous and donor HLMC (left panel); indigenous origin (central panel); donor HLMC origin (right panel). n = 5 per group. *P < 0.05 versus the same group at day 0; +P < 0.05 versus group CON at the corresponding time point. (c–g) Examples of chimerism and proliferation (3H‐thymidine labelling) of GFP+ HLMC‐derived PHA‐L‐stained cells at 3 days after HgCl2. (c) Black arrowheads indicate PHA‐L stained (blue colour in tubular cell apical membrane) donor GFP+ HLMC‐derived tubular cells (brown colour in cytoplasm) under bright field, and black arrows indicate 3H‐thymidine labelling of PHA‐L‐stained HLMC‐derived tubular cells, ×500. (d) The same field under dark field, white arrowheads point to silver grains (3H‐thymidine labelling) and white arrows indicate 3H‐thymidine labelling of PHA‐L‐stained HLMC‐derived tubular cells (×500). (e) Bright field and (f) dark field are the higher magnification of yellow box area in (c). (g) The images in (e) and (f) were combined to help to show silver grains over cells that are donor GFP+ HLMC‐derived PHA‐L‐stained tubular cells. GFP, green fluorescent protein; HLMC, haematopoietic lineage marrow cell; PHA‐L, Phaseolus vulgaris leucoagglutinin.

Article Snippet: After GFP staining, tissue sections were microwaved in 10 m m tri‐sodium citrate (pH 6.0) for 10 min and biotin was blocked using a biotin blocking kit (DAKO), and then incubated for 45 min with biotinylated Phaseolus vulgaris leucoagglutinin (PHA‐L) 1/100 (B‐1115, Vector Laboratories, Burlingame, CA, USA) to detect proximal convoluted tubules, or biotinylated peanut agglutinin (PNA) 1/800 (B‐1075, Vector Laboratories) to detect distal convoluted tubules.

Techniques: Staining, Derivative Assay, Cell Counting

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: Journal of molecular endocrinology

Article Title: Stomach gastrin is regulated by sodium via PPAR-α and dopamine D 1 receptor

doi: 10.1530/JME-19-0053

Figure Lengend Snippet: Human stomach antrum and G cell immunochemistry for gastrin and PHA-L. (A) Gastrin, phytohemagglutinin-leucoagglutinin (PHA-L, marker of complex type carbohydrate residues which are expressed in gastric parietal cells), and nuclei (blue) in the antrum of fresh human stomach. Scale bar = 10 μm. (B) Gastrin and PHA-L staining in G cells isolated from human stomach and gastrin staining in SW626 cells (ovarian adenocarcinoma cells). G cell- and SW626 cell-control using non-specific rabbit IgG antibody show almost no staining. NG = negative control, non-specific rabbit IgG antibody. Scale bar = 10 μm.

Article Snippet: The cells were then stained with anti-goat gastrin antibody (Santa Cruz) or anti-rabbit antibodies to various targets (gastrin, DAKO 1:200), complex type carbohydrate residues marker Phaseolus vulgaris -leucoagglutinin (PHA-L, Vector Laboratories), D 1 R (generated in our laboratory) ( Sanada et al. 1999 ), D 5 R (Santa Cruz, 1:100), tyrosine hydroxylase (TH, Cell Signaling, 1:200), and DOPA decarboxylase (DDC, Millipore, 1:200) for 2 h at room temperature.

Techniques: Marker, Staining, Isolation, Negative Control

Journal: BioTechniques

Article Title: Isolation of urinary extracellular vesicles from Tamm- Horsfall protein-depleted urine and their application in the development of a lectin-exosome-binding assay.

doi: 10.2144/000114208

Figure Lengend Snippet: Figure 4. Lectin binding pattern of urinary EVs from healthy subjects. Immobilized uEVs were allowed to react with biotinylated plant lectins for 30 min at RT. The unbound material was washed out, followed by addition of avidin/biotinylated HRPO and TMB solution. Optical density was monitored at 450 nm. Binding of each lectin was normalized to binding of anti-CD63 antibody, which was run as the control for uEV immobilization. The results are mean values of three experiments. uEVs from both male and female donors were analyzed. Error bars represent the standard deviation. Lectins: Con A (specific for high mannose-type, hybrid-type, and biantennary complex type N-glycans), WGA (specific for GlcNAcb1.4 oligomers/Galb1,4GlcNAc), RCA (specific for Galb1,4GlcNAc), SNA (specific for sialic acid a2,6-Gal), MAA (specific for sialic acid a2,3-Gal), PHA-E (specific for bisected biantennary chains), PHA-L (specific for tri/tetraantennary complex-type N-glycans), LCA (specific for bi/triantennary complex-type with core fucose), PNA (specific for Galb1,3GalNAca-Ser/Thr, T antigen), VVL (specific for terminal GalNAca-Ser/Thr, Tn antigen/ GalNAc1,3Gal), DBL (specific for a1,3GalNAc and blood group A antigen).

Article Snippet: Biotinylated plant lectins [concanavalin A (Con A), Lens culinaris agglutinin (LCA), peanut agglutinin (PNA), Sambucus nigra agglutinin (SNA), Maackia amurensis lectin II (MAL II), wheat germ agglutinin (WGA), Ricinus communis agglutinin I (RCA I), Vicia villosa lectin (VVL), and Dolichos biflorus agglutinin (DBA)], VECTASTAIN ABC Kit, biotinylated goat anti-mouse IgG, and biotinylated rabbit anti-goat IgG were purchased from Vector Laboratories (Burlingame, CA).

Techniques: Binding Assay, Avidin-Biotin Assay, Control, Standard Deviation

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