Journal: Biomedicines

Article Title: Characterization of Mesothelin Glycosylation in Pancreatic Cancer: Decreased Core Fucosylated Glycoforms in Pancreatic Cancer Patients’ Sera

doi: 10.3390/biomedicines10081942

Figure Lengend Snippet: Lectins used in WB.

Article Snippet: Fluorescein labelled Phaseolus vulgaris Erythroagglutinin (PHA-E) , Galβ4GlcNAcβ2Manα6 (GlcNAcβ4) (GlcNAcβ4Manα3) Manβ4 , 2 µg/mL , FL-1121 (Vector Laboratories).

Techniques: Concentration Assay, Plasmid Preparation

Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells and endothelial progenitor cells decrease renal injury in experimental swine renal artery stenosis through different mechanisms

doi: 10.1002/stem.1263

Figure Lengend Snippet: Top: Representative images of CM-DiI labeled (red) endothelial progenitor cells (EPC) or mesenchymal stem cells (MSC) in the post-stenotic kidneys of pigs with renal artery stenosis (RAS) 4 weeks after cell delivery. Green shows peanut agglutinin (PA, green arrow), a distal tubular marker, and cyan shows a proximal tubular marker phaseolus vulgaris erythroagglutinin (PHA-E, cyan arrow). EPC showed mainly tubular engraftment (yellow arrow), while MSC tend to integrate into both proximal tubules (yellow arrow) and interstitial area (red arrow). Bottom: Both EPC and MSC improved RBF and GFR in pigs with RAS, yet MSC more effectively restored GFR. *p<0.05 vs. Normal, †p<0.05 vs. RAS. Scale bar=200µm

Article Snippet: Furthermore, frozen kidney sections from pigs infused with cells were stained with the distal tubular marker peanut agglutinin (5ug/ml, Vector) and proximal tubular marker phaseolus vulgaris erythroagglutinin (PHA-E, 5ug/ml, Vector).

Techniques: Labeling, Marker

Journal: bioRxiv

Article Title: Overexpression of CD47 is associated with brain overgrowth in 16p11.2 deletion syndrome

doi: 10.1101/808022

Figure Lengend Snippet: A. Representative histograms show the mean fluorescence intensities (MFI) of cell-surface expression of Calreticulin (CRT) in NPCs derived from control, 16p_del, and 16p_dup lines. B. Quantification of CRT MFI of cell-surface expression of CRT in the 16p_del NPCs relative to 16p_dup and control NPCs. C. Quantification of Phaseolus Vulgaris Leucoagglutinin (PHA-L), indicative of asialoglycan binding sites for CRT, in NPCs in 16p_deletion lines relative to control and 16p_duplication lines. D. Representative histograms show the MFI of cell-surface expression of CRT in OPCs derived from control, 16p_del, and 16p_dup lines. E. Quantification of CRT MFI of cell-surface expression of CRT in the 16p_del OPCs relative to 16p_dup and control OPCs. F. Quantification of PHA-L in OPCs in 16p_deletion lines relative to control and 16p_duplication lines. n=2 biological replicates per cell line (16p_dup, n=2; control, n=3; 16p_del, n=4 cell lines). All data are mean ± SE; P values were determined by one-way ANOVA followed by post-hoc Tukey HSD Test.

Article Snippet: The lectin fluorescein labeled Phaseolus Vulgaris Leucoagglutinin (PHA-L) (1:200; Vector Laboratories) was used to stain for PHA-L. For O4 staining, the cells were first incubated in primary antibody mouse-anti-O4 antibody (Clone 81; 1:100; EMD millipore), followed by secondary antibody incubation, anti-Mouse IgM Alexa Fluor 488 (1:1000; Life Technologies).

Techniques: Fluorescence, Expressing, Derivative Assay, Binding Assay

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