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: Proteomics

Article Title: Focused glycomic analysis of the N -linked glycan biosynthetic pathway in ovarian cancer

doi: 10.1002/pmic.200800157

Figure Lengend Snippet: Lectin blot analysis of glycoproteins from mouse and human endometrioid ovarian carcinoma. (A) Glycoproteins extracted from mouse endometrioid ovarian tumors (lanes 2, 4, 6, and 8) or normal mouse ovary (lanes 1, 3, 5, and 7) that were nonadherent to Con A were separated on 4–12% Bis-Tris gels before transfer to PVDF membrane and detection using biotinylated lectins and streptavidin–HRP. Panel below shows the densitometry analysis of bands from normal (NL) or ovarian tumor (OT) in the 49–250 kDa range from the blots shown above with normal set at 1.0 for comparison. Fold increase was adjusted for lectin pull-down inputs based on the levels of ERK2 on a 10% input blot (data not shown). (B) Glycoproteins nonadherent to Con A from human endometrioid ovarian cancer cases (711, 741, and 471) and normal human ovary (NL) were separated on 4–12% Bis-Tris gels before lectin blot detection as described. The panel below represents the densitometry results for glycoproteins 49–250 kDa relative to normal set at 1.0. Increases relative to normal were adjusted for input using ERK2 analysis form 10% input blots (data not shown).

Article Snippet: Unbound fractions, 10 µg, were separated on 4–12 % NuPage Bis Tris gels and transferred to PVDF membrane at 25 V for 1.5 h. Membranes were blocked overnight in 3% BSA/TBST buffer before lectin blot detection using a 1:5000 dilution of the following biotinylated lectins: ( Phaseolus vulgaris leucoagglutinin (L-PHA), P. vulgaris erythroagglutinin (E-PHA), Aleuria aurantia (AAL), and Datura stramonium (DSL), Vector Labs).

Techniques: