Journal: bioRxiv

Article Title: Differential Expression of N- and O-glycans on HeLa Cells-Revealed by Direct Fluorescent Glycan Labeling with Recombinant Sialyltransferases

doi: 10.1101/580571

Figure Lengend Snippet: Fetal bovine fetuin (F) and asialofetuin (AF) were labeled by various recombinant sialyltransferases with indicated fluorophore conjugated CMP-sialic acids. The labeling reactions were separated on SDS-PAGE and imaged by trichloroethanol (TCE) staining and fluorescent imager. A, B and C are for a same gel to test the tolerance of Alexa Fluor® 555 by the indicated enzymes. A is a TCE image. B and C are fluorescent images with different contrast. D E and F are for a same gel to test the tolerance of Alexa Fluor® 488, Alexa Fluor® 555 and Cy5 by the indicated enzymes. D is a TCE image. E and F are fluorescent images with different contrast. 31, ST3Gal1; 32, ST3Gal2; 34, ST3Gal4; 61, ST6Gal1; 6A1, ST6GalNAc1; 6A2, ST6GalNAc2; 6A4, ST6GalNAc4. Same amount of protein (2.5 μg) was loaded into each lane in both A and D ; however, due to the presence of multiple benzene rings in Alexa-Fluor® dyes, Alexa Fluor® 488 and 555 labeled proteins showed significantly increased band intensities in TCE gels. Labeling was success in the presence of C.P neuraminidase in panel A , suggesting that the neuraminidase has no glycosidase activity on the introduced fluorophore conjugated sialic acids. MM, molecular marker.

Article Snippet: CMP-Azido-Sialic acid, UDP-Azido-GlcNAc, Biotinylated Alkyne, DAPI, recombinant human ST3Gal1, ST3Gal2, ST3Gal4, ST6Gal1, ST6GalNAc1, ST6GalNAc4, OGT and C. perfringens Neuraminidase were from Bio-Techne.

Techniques: Labeling, Recombinant, SDS Page, Staining, Activity Assay, Marker

Journal: bioRxiv

Article Title: Differential Expression of N- and O-glycans on HeLa Cells-Revealed by Direct Fluorescent Glycan Labeling with Recombinant Sialyltransferases

doi: 10.1101/580571

Figure Lengend Snippet: Confluent HeLa cells were imaged by ST3Gal1 ( A and B ), ST6GalNAc4 ( C and D ), ST6Gal1 ( E and F ) before ( A, C, E ) and after ( B, D, F ) C.p. neuraminidase treatment. The cells were stained by ST3Gal1 and ST6Gal1 only after C. perfringens neuraminidase treatment, suggesting that both Core-1 O-glycan (T antigen) and N-glycan were highly sialylated on these cells. On the contrary, sT antigen was only stained by ST6GalNAc4 prior to C. p neuraminidase treatment ( C ). While sT antigen revealed in panels B and C evenly distributed on cell surface, N-glycans revealed in panel F showed a more peripheral localization. Additionally, not all cells in panel B and C were equally positive for sT antigen. All images were revealed by Alexa Fluor® 555. For comparison, intracellular contents were stained by OGT in H using UDP-azido-GlcNAc that was further reacted to biotin-alkyne and revealed by streptavidin conjugated Alexa Fluor® 555. Nuclei were stained in blue with DAPI.

Article Snippet: CMP-Azido-Sialic acid, UDP-Azido-GlcNAc, Biotinylated Alkyne, DAPI, recombinant human ST3Gal1, ST3Gal2, ST3Gal4, ST6Gal1, ST6GalNAc1, ST6GalNAc4, OGT and C. perfringens Neuraminidase were from Bio-Techne.

Techniques: Staining, Comparison

Journal: bioRxiv

Article Title: Differential Expression of N- and O-glycans on HeLa Cells-Revealed by Direct Fluorescent Glycan Labeling with Recombinant Sialyltransferases

doi: 10.1101/580571

Figure Lengend Snippet: HeLa cells at sub-confluence ( A and B ) and fully confluence ( C and D ) conditions were briefly treated with C. perfringens neuraminidase to remove preexisting sialic acids and then imaged for Core-1 O-glycans by ST3Gal1 using Alexa Fluor® 555 conjugated CMP-SA (red) and N-glycans by ST6Gal1 using Alexa Fluor® 488 conjugated CMP-SA (green). A and B are for the exact same viewing area. C and D are for the exact same viewing area. Cell nuclei were revealed with DAPI staining in blue in B and D . N-glycans are expressed preferentially at peripheral positions, especially on protrusions of the cells, compared to Core-1 O-Glycans that are more centralized and evenly distributed on cells.

Article Snippet: CMP-Azido-Sialic acid, UDP-Azido-GlcNAc, Biotinylated Alkyne, DAPI, recombinant human ST3Gal1, ST3Gal2, ST3Gal4, ST6Gal1, ST6GalNAc1, ST6GalNAc4, OGT and C. perfringens Neuraminidase were from Bio-Techne.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans

doi: 10.3389/fimmu.2018.02857

Figure Lengend Snippet: Expression of PNA-reactive glycans by germinal center B cells is correlated with downregulation of the α2,3-sialyltransferase ST3Gal1. (A) Gating strategy for analyzing tonsillar naïve (N), germinal center (GC), memory (M), and plasmablast (PB) B cells by flow cytometry. Representative fluorescence minus one (FMO) controls used for gating of CD27 are shown. (B) Analysis of peanut lectin binding to human tonsil B cells. Representative histograms of results are shown ( left ) as well as quantification of geometric mean fluorescence intensities (MFI) ( right ). (C) Schematic of synthesis of potential PNA-reactive O-linked glycans on B cells. O-glycan synthesis is initiated in the Golgi apparatus by polypeptide N-acetylgalactosamine transferases ( GALNTs ), which transfer a single GalNAc to select serine/threonine residues of a polypeptide backbone. The initiating GalNAc can be terminally sialylated, or further extended by C1GalT1 ( C1GALT1 ) to form the simplest PNA-reactive epitope, a Core 1 O-glycan termed “T-antigen.” This core T-antigen moiety can be branched and elongated by other glycosyltransferases to form extended Core 2 O-glycans, which retain binding to PNA, or modified with sialic acid by the α2,3-sialyltransferase ST3Gal1 ( ST3GAL1 ), which destroys PNA reactivity. Endogenous (or exogenous) sialidases may remove sialic acids and restore PNA binding. (D) Analysis of ST3GAL1 expression in tonsillar B cells by quantitative real-time reverse transcription PCR (qRT-PCR), sorted as in (A) . Data are normalized to the housekeeping gene VCP and presented relative to naïve B cells. Data are representative of eight (B) or three (D) distinct tonsil specimens pooled from two (B) or three (D) independent experiments. Statistics were calculated using a Kruskal–Wallis test with Dunn's multiple comparisons test (B) or One-way analysis of variance (ANOVA) and Tukey's multiple comparisons test. Throughout, bars and error bars depict the mean and SEM, respectively. ns = not significant, *** p ≤ 0.001. ΔMFI, background subtracted geometric mean fluorescence intensity; GalNAc, N-acetylgalactosamine; Gal, galactose; Sia, sialic acid.

Article Snippet: To generate ST3Gal1 overexpression Ramos B cells, human ST3Gal1 cDNA (Origene #SC111017) was amplified by PCR and then subcloned into pLVX-EF1α-IRES-ZsGreen1 (Clontech #631982), a bicistronic lentiviral expression vector allowing for simultaneous co-expression of ST3Gal1 and ZsGreen1 from a single mRNA transcript.

Techniques: Expressing, Flow Cytometry, Fluorescence, Binding Assay, Modification, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans

doi: 10.3389/fimmu.2018.02857

Figure Lengend Snippet: ST3Gal1 regulates PNA binding in B cells by sialylating Core 1 O-glycans. (A) Validation of ST3GAL1 overexpression in Ramos B cells by qRT-PCR. Data were normalized to housekeeping control VCP and presented relative to vector control. (B) Representative histogram ( left ) and quantification ( right ) of flow cytometric analysis of PNA binding to vector control and ST3Gal1OE Ramos B cells. The Core 1 O-glycan/ T-antigen specificity of PNA is depicted at top right. (C) Representative histogram ( left ) and quantification ( right ) of flow cytometric analysis of MAL-II plant lectin binding to vector control and ST3Gal1OE Ramos B cells. The α2,3-sialylated Core 1 O-glycan/sialylated T-antigen glycan favored by MAL-II lectin is depicted at top right. (D) Representative histogram ( left ) and quantification ( right ) of PNA binding to vector control ( top ) or ST3Gal1OE Ramos B cells ( bottom) before and after removal of sialic acids by intact cell treatment with Arthrobacter ureafaciens sialidase. (E) Representative histogram ( left ) and quantification ( right ) of MAL-II binding to vector control ( top ) or ST3Gal1OE ( bottom) Ramos B cells before and after sialidase treatment, as in (D) . Data in (B–E) are from three independent experiments with three biological replicates in total. Statistics were calculated using Welch's unpaired, two-tailed t -test (B–E) . Throughout, bars and error bars depict the mean and SEM, respectively. ns = not significant, * p ≤ 0.05, ** p < 0.01, ΔMFI, background subtracted geometric mean fluorescence intensity; Ctrl, vector control. ST3OE; ST3Gal1 overexpression.

Article Snippet: To generate ST3Gal1 overexpression Ramos B cells, human ST3Gal1 cDNA (Origene #SC111017) was amplified by PCR and then subcloned into pLVX-EF1α-IRES-ZsGreen1 (Clontech #631982), a bicistronic lentiviral expression vector allowing for simultaneous co-expression of ST3Gal1 and ZsGreen1 from a single mRNA transcript.

Techniques: Binding Assay, Over Expression, Quantitative RT-PCR, Plasmid Preparation, Two Tailed Test, Fluorescence

Journal: Frontiers in Immunology

Article Title: Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans

doi: 10.3389/fimmu.2018.02857

Figure Lengend Snippet: Overexpression of ST3Gal1 in B cells modulates binding of glycosylation sensitive CD45 antibodies. (A) Representative histogram ( left ) and quantification ( right ) of binding of two glycosylation-sensitive CD45 antibodies (MEM55 and B220) and one glycosylation-insensitive antibody (total CD45, clone HI30) to vector control and ST3Gal1OE Ramos B cells by flow cytometry. (B) Western blot analysis of binding of CD45 mAb MEM55 (800 nm fluorescence channel) and CD45 mAb HI30 (700 nm fluorescence channel) to Ramos vector control and ST3Gal1OE lysates. (C) Representative histograms depicting flow cytometric analysis of CD45 antibody binding to vector control and ST3Gal1OE Ramos B cells, before and after treatment with Arthrobacter ureafaciens sialidase. Data in (A,C) are from three independent experiments with three biological replicates. Data in (B,C) are representative of similar results from three (B) or two (C) independent experiments. Statistics in (A) were calculated using Welch's unpaired, two-tailed t -test. Throughout, bars and error bars depict the mean and SEM, respectively. ns, not significant, ** p ≤ 0.01, *** p ≤ 0.001, ΔMFI, background subtracted geometric mean fluorescence intensity; Ctrl, vector control; ST3OE, ST3Gal1OE.

Article Snippet: To generate ST3Gal1 overexpression Ramos B cells, human ST3Gal1 cDNA (Origene #SC111017) was amplified by PCR and then subcloned into pLVX-EF1α-IRES-ZsGreen1 (Clontech #631982), a bicistronic lentiviral expression vector allowing for simultaneous co-expression of ST3Gal1 and ZsGreen1 from a single mRNA transcript.

Techniques: Over Expression, Binding Assay, Plasmid Preparation, Flow Cytometry, Western Blot, Fluorescence, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans

doi: 10.3389/fimmu.2018.02857

Figure Lengend Snippet: Overexpression of ST3Gal1 truncates O-glycans in B cells. (A) Representative histograms ( left ) and quantification ( right ) of binding of the Core 2 poly-LacNAc binding lectin Solanum tuberosum agglutinin (STA) to vector control and ST3Gal1OE Ramos B cells by flow cytometry. (B) Representative histograms ( left ) and quantification ( right ) of binding of the CD43 mAb 1D4 (Core 2 O-glycan-specific glycoform) to vector control and ST3Gal1OE Ramos B cells by flow cytometry. (C) Cellular O-glycome Reporter/Analysis (CORA) of untransduced Ramos, Ramos vector control, and Ramos ST3Gal1OE B cells. Depicted are MALDI-TOF MS spectra of peracetylated Benzyl-α-GalNAc-linked O-glycans. Structures above a bracket have not been unequivocally defined. Indicated areas in the spectra have a 20-fold magnification. “M” and “m” designations indicate major and minor abundances, respectively. Cartoon structures were drawn according to http://www.functionalglycomics.org guidelines and are representative from repeat experiments on two different biological replicates. Structure assignments are based on composition, tandem mass spectrometry, and biosynthetic knowledge. Full methods for MS analysis can be found in Materials and Methods. Data depict results from three (A,B) or two (C) biological replicates. Statistics in (A) and (B) were calculated using Welch's unpaired, two-tailed t -test. Throughout, bars and error bars depict the mean and SEM, respectively. ** p ≤ 0.01, ΔMFI, background subtracted geometric mean fluorescence intensity. Ctrl, vector control; ST3OE, ST3Gal1OE; Fuc, fucose; Man, mannose; Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; NeuAc, N-acetylneuraminic acid (sialic acid).

Article Snippet: To generate ST3Gal1 overexpression Ramos B cells, human ST3Gal1 cDNA (Origene #SC111017) was amplified by PCR and then subcloned into pLVX-EF1α-IRES-ZsGreen1 (Clontech #631982), a bicistronic lentiviral expression vector allowing for simultaneous co-expression of ST3Gal1 and ZsGreen1 from a single mRNA transcript.

Techniques: Over Expression, Binding Assay, Plasmid Preparation, Flow Cytometry, Mass Spectrometry, Two Tailed Test, Fluorescence

Journal: Cell Death & Disease

Article Title: Sialyltransferase ST3GAL1 promotes cell migration, invasion, and TGF-β1-induced EMT and confers paclitaxel resistance in ovarian cancer

doi: 10.1038/s41419-018-1101-0

Figure Lengend Snippet: ST3GAL1 expression and clinicopathological characteristics of 78 patients with ovarian cancer

Article Snippet: Primary antibodies included a Rabbit Polyclonal antibody against human ST3GAL1 (LS-C185763, LifeSpan BioSciences) at a dilution of 1:1000.

Techniques: Expressing, Histopathology

Journal: Cell Death & Disease

Article Title: Sialyltransferase ST3GAL1 promotes cell migration, invasion, and TGF-β1-induced EMT and confers paclitaxel resistance in ovarian cancer

doi: 10.1038/s41419-018-1101-0

Figure Lengend Snippet: a ST3GAL1 expression in 78 ovarian cancer tumor tissues and 15 relative normal tissues analyzed by qRT-PCR (**p < 0.01, Student’s t-test). b ST3GAL1 protein expression was analyzed by western blots in ovarian cancer tissues. c ST3GAL1 expression analyzed by immunohistochemistry in ovarian cancer tissues and normal tissues, respectively

Article Snippet: Primary antibodies included a Rabbit Polyclonal antibody against human ST3GAL1 (LS-C185763, LifeSpan BioSciences) at a dilution of 1:1000.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Sialyltransferase ST3GAL1 promotes cell migration, invasion, and TGF-β1-induced EMT and confers paclitaxel resistance in ovarian cancer

doi: 10.1038/s41419-018-1101-0

Figure Lengend Snippet: a qRT-PCR analysis of ST3GAL1 expression in the ovarian cancer cell lines SKOV-3, OVCAR3, and A2780 and the normal ovarian cell line NOEC (*p < 0.05, **p < 0.01, and ***p < 0.001, one-way ANOVA). b Knockdown of ST3GAL1 (ST3GAL1 shRNA1#–3#) in SKOV-3 cells and qRT-PCR assessment of ST3GAL1 expression (*p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA). SKOV-3 cells were transfected with ST3GAL1-shRNA1# or shRNA2# and A2780 cells were transfected overexpression ST3GAL1 (ST3GAL1-OE). c–e Cell growth was determined by the CCK-8 assay (*p < 0.05, **p < 0.01, one-way ANOVA). f, g Transwell assay was used to detect cell migration and invasion (*p < 0.05, and **p < 0.01, one-way ANOVA)

Article Snippet: Primary antibodies included a Rabbit Polyclonal antibody against human ST3GAL1 (LS-C185763, LifeSpan BioSciences) at a dilution of 1:1000.

Techniques: Quantitative RT-PCR, Expressing, Knockdown, Transfection, Over Expression, CCK-8 Assay, Transwell Assay, Migration

Journal: Cell Death & Disease

Article Title: Sialyltransferase ST3GAL1 promotes cell migration, invasion, and TGF-β1-induced EMT and confers paclitaxel resistance in ovarian cancer

doi: 10.1038/s41419-018-1101-0

Figure Lengend Snippet: a, b SKOV-3 and A2780 cells were treated with or without 10 ng/mL TGF-β1, qRT-PCR analyzed ST3GAL1 mRNA expression (**p < 0.01, Student’s t-test). SKOV-3 cells treated with TGF-β1 or transfected with ST3GAL1-shRNA1# or shRNA2#, or both TGF-β1 and ST3GAL1-shRNA1# or shRNA2#, untransfected cells were used as a control. c, d E-cadherin, N-cadherin, and Vimentin protein expression were analyzed by western blots. A2780 cells treated with TGF-β1 or transfected with ST3GAL1-OE, or both TGF-β1 and ST3GAL1-OE, untransfected cells were used as a control. e, f E-cadherin, N-cadherin, and Vimentin protein expression were analyzed by western blots. (*p < 0.05, **p < 0.01, one-way ANOVA)

Article Snippet: Primary antibodies included a Rabbit Polyclonal antibody against human ST3GAL1 (LS-C185763, LifeSpan BioSciences) at a dilution of 1:1000.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Control, Western Blot

Journal: Cell Death & Disease

Article Title: Sialyltransferase ST3GAL1 promotes cell migration, invasion, and TGF-β1-induced EMT and confers paclitaxel resistance in ovarian cancer

doi: 10.1038/s41419-018-1101-0

Figure Lengend Snippet: SKOV-3 cells treated with TGF-β1 or transfected with ST3GAL1-shRNA1# or shRNA2#, or both TGF-β1 and ST3GAL1-shRNA1# or shRNA2#, untransfected cells were used as a control. A2780 cells treated with TGF-β1 or transfected with ST3GAL1-OE, or both TGF-β1 and ST3GAL1-OE, untransfected cells were used as a control. a, b E-cadherin and N-cadherin protein expression were analyzed by cell immunofluorescence

Article Snippet: Primary antibodies included a Rabbit Polyclonal antibody against human ST3GAL1 (LS-C185763, LifeSpan BioSciences) at a dilution of 1:1000.

Techniques: Transfection, Control, Expressing, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Sialyltransferase ST3GAL1 promotes cell migration, invasion, and TGF-β1-induced EMT and confers paclitaxel resistance in ovarian cancer

doi: 10.1038/s41419-018-1101-0

Figure Lengend Snippet: SKOV-3 cells treated with TGF-β1 or transfected with ST3GAL1-shRNA1# or shRNA2#, or both TGF-β1 and ST3GAL1-shRNA1# or shRNA2#, untransfected cells were used as a control. A2780 cells treated with TGF-β1 or transfected with ST3GAL1-OE, or both TGF-β1 and ST3GAL1-OE, untransfected cells were used as a control. a, b SKOV-3 and A2780 cells migration and invasion were analyzed by Transwell assay. *p < 0.05, and **p < 0.01

Article Snippet: Primary antibodies included a Rabbit Polyclonal antibody against human ST3GAL1 (LS-C185763, LifeSpan BioSciences) at a dilution of 1:1000.

Techniques: Transfection, Control, Migration, Transwell Assay

Journal: Cell Death & Disease

Article Title: Sialyltransferase ST3GAL1 promotes cell migration, invasion, and TGF-β1-induced EMT and confers paclitaxel resistance in ovarian cancer

doi: 10.1038/s41419-018-1101-0

Figure Lengend Snippet: a, b SKOV-3 and A2780 cells were treated with 0, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400, and 800 ng/mL Taxol for 7 days, and the half maximal inhibitory concentration (IC 50) was detected. The IC50 of A2780 is 101 ng/mL and the IC50 of SKOV-3 cells is 147 ng/mL. c, d A colony formation assay of A2780 cells transfected with ST3GAL1 (ST3GAL1-OE) for overexpression. SKOV-3 cells transduced with shRNA1# or 2# for ST3GAL1 knockdown. Cells were grown for 14 days under treatment with or without 101 ng/mL Taxol for A2780 cells or 147 ng/mL Taxol for SKOV-3 cells and stained with crystal violet. The colonies were counted and captured. The data represent the mean ± SD from three independent experiments. *p < 0.05, and **p < 0.01

Article Snippet: Primary antibodies included a Rabbit Polyclonal antibody against human ST3GAL1 (LS-C185763, LifeSpan BioSciences) at a dilution of 1:1000.

Techniques: Concentration Assay, Colony Assay, Transfection, Over Expression, Transduction, Knockdown, Staining

Journal: Cell Death & Disease

Article Title: Sialyltransferase ST3GAL1 promotes cell migration, invasion, and TGF-β1-induced EMT and confers paclitaxel resistance in ovarian cancer

doi: 10.1038/s41419-018-1101-0

Figure Lengend Snippet: a The tumor growth curve of overexpression ST3GAL1 A2780 cells was compared with vector-expressing cells after Taxol treatment. Tumor growth was assessed in nude mice that were subcutaneously injected in the right flank with 2.0 × 106 stable transfectants. b, c Average tumor weight was measured and growth curves were generated (N = 6, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA). d Ki67 expression in the four groups was detected by immunohistochemistry, and TUNEL analysis in the four groups

Article Snippet: Primary antibodies included a Rabbit Polyclonal antibody against human ST3GAL1 (LS-C185763, LifeSpan BioSciences) at a dilution of 1:1000.

Techniques: Over Expression, Plasmid Preparation, Expressing, Injection, Generated, Immunohistochemistry, TUNEL Assay