Journal: PLoS ONE

Article Title: mAb Das-1 recognizes 3’-Sulfated Lewis A/C, which is aberrantly expressed during metaplastic and oncogenic transformation of several gastrointestinal Epithelia

doi: 10.1371/journal.pone.0261082

Figure Lengend Snippet: Chemical deglycosylation of the antigen results in near complete loss of signal as measured by western blot analysis using ( A ) Das-1 IgM and ( B ) Das-1 IgG. Quantification of band intensity is presented below each band.

Article Snippet: Das-1 IgM or IgG were used at 1 μg/mL and Goat anti-Mouse IgM (LiCOR 926–32280) or Donkey Anti-Mouse IgG (LiCOR 926–32212) were used at 1:5000 and imaged on a Odyssey CLX.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: mAb Das-1 recognizes 3’-Sulfated Lewis A/C, which is aberrantly expressed during metaplastic and oncogenic transformation of several gastrointestinal Epithelia

doi: 10.1371/journal.pone.0261082

Figure Lengend Snippet: Das-1 IgG (at 5 & 50 μg/mL) and Das-1 IgM (at 5 μg/mL) are plotted in A, B, and C respectively as the average relative fluorescence units (of 6 technical replicates) plus/minus standard deviation. The top 9 glycans for each arrays are listed to the right of each figure. Colored arrows emphasize that Das-1 IgG and Das-1 IgM recognize the same set of glycans. The complete data sets are provided in (5 μg/mL IgG), (50 μg/mL IgG), and (5 μg/mL IgM) and are available for download on the Consortium for Functional Glycomics website ( www.functionalglycomics.org ).

Article Snippet: Das-1 IgM or IgG were used at 1 μg/mL and Goat anti-Mouse IgM (LiCOR 926–32280) or Donkey Anti-Mouse IgG (LiCOR 926–32212) were used at 1:5000 and imaged on a Odyssey CLX.

Techniques: Fluorescence, Standard Deviation, Functional Assay

Journal: PLoS ONE

Article Title: mAb Das-1 recognizes 3’-Sulfated Lewis A/C, which is aberrantly expressed during metaplastic and oncogenic transformation of several gastrointestinal Epithelia

doi: 10.1371/journal.pone.0261082

Figure Lengend Snippet: Direct ELISA using Das-1 IgG ( A ) or Das-1 IgM ( C ) in the absence or presence of several free glycans in solution at 200 μM. Direct ELISA using Das-1 IgG ( B ) or Das-1 IgM ( D ) against a titration series of 3’-Sulfo-Le A . Data reported as average ± standard deviation of three technical replicates normalized to the reaction without glycans (PBS). Key : Schematic diagram of the relevant Lewis antigens is provided for reference.

Article Snippet: Das-1 IgM or IgG were used at 1 μg/mL and Goat anti-Mouse IgM (LiCOR 926–32280) or Donkey Anti-Mouse IgG (LiCOR 926–32212) were used at 1:5000 and imaged on a Odyssey CLX.

Techniques: Direct ELISA, Titration, Standard Deviation

Journal: Scientific Reports

Article Title: Physico-chemical characterization of bilirubin-10-sulfonate and comparison of its acid–base behavior with unconjugated bilirubin

doi: 10.1038/s41598-021-92377-8

Figure Lengend Snippet: Dependence of effective electrophoretic mobility of ( a ) bilirubin-10-sulfonate (ranarubin) and ( b ) UCB. Background electrolytes 1–3, 6, 9–13 were used for the bilirubin-10-sulfonate studies; background electrolytes 3–5, 7, 8 for the UCB studies. Depicted points are the mean ± SD values of three experimental values. The position of the inflection points was determined using the numerical interpolation procedure using the standard Boltzmann sigmoidal function defined in the Origin software (Origin (Pro), Version 2016, OriginLab Corporation, Northampton, MA, USA Corporation, MA USA).

Article Snippet: The position of the inflection points was determined using the numerical interpolation procedure using the standard Boltzmann sigmoidal function defined in the Origin software (Origin (Pro), Version 2016, OriginLab Corporation, Northampton, MA, USA Corporation, MA USA).

Techniques: Software

Journal: Reproduction

Article Title: Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

doi: 10.1530/rep-18-0333

Figure Lengend Snippet: Figure 1 mRNA expression pattern of versican in human endometrium. (A) RT-PCR analysis shows that all versican isoforms (V0–V3) are detected in human endometrial tissue throughout the menstrual cycle. (B) Expression of versican (a) V0, (b) V1, (c) V2 and (d) V3 across different phases of the menstrual cycle was analyzed by quantitative RT-PCR (proliferative, n = 4; early secretory, n = 4; mid-secretory, n = 4; late secretory, n = 4). One-way ANOVA was used for statistical analysis.

Article Snippet: Goat anti-human versican polyclonal antibody (pAb; #AF3054), which recognizes an amino acid sequence in the GAG-β domain specific to the V0 and V1 isoforms, was purchased from R&D Systems.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Reproduction

Article Title: Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

doi: 10.1530/rep-18-0333

Figure Lengend Snippet: Figure 2 Versican protein expression in human endometrium. (A) Endometrial tissues of various menstrual cycle phases were immunostained with anti-versican polyclonal antibody specific for the V0 and V1 isoforms. (a) Early proliferative phase, (b) mid- proliferative phase, (c) late proliferative phase, (d) early secretory phase, (e) mid-secretory phase, (f) late secretory phase; (g)–(l) are higher magnification views of the area indicated in (a)–(f), respectively. (m) Glandular epithelium in the functional layer of mid-secretory endometrium. (n) Negative control for (m) in which the anti-versican antibody was replaced by isotype-matched control antibody. Versican expression was detected in both endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs) throughout the menstrual cycle. The intensity of EEC versican expression was higher in the secretory phases than in the proliferative phases. Note that intense versican expression was also detected in intra-glandular products secreted from EECs and are indicated by arrowheads in (m). Scale bars = 100 µm in (a)–(f); 50 μm in (g)–(n). (B) Signal intensities of versican V1 were assessed in the immunohistochemical images of whole endometria of various menstrual cycle phases (representative images shown in A). Immunointensity scores (0, negative; 1, very weak; 2, weak; 3, moderate; 4, strong; 5, very strong) were separately given to (a) EECs and (b) ESCs. Note that immunointensity scores of (a) EECs were significantly higher in the secretory phases than in the proliferative phases, whereas those of (b) ESCs were constant throughout the menstrual cycle. In all panels, error bars represent the standard deviation. *P < 0.05; **P < 0.01; N.S., not significant.

Article Snippet: Goat anti-human versican polyclonal antibody (pAb; #AF3054), which recognizes an amino acid sequence in the GAG-β domain specific to the V0 and V1 isoforms, was purchased from R&D Systems.

Techniques: Expressing, Functional Assay, Negative Control, Control, Immunohistochemical staining, Standard Deviation

Journal: Reproduction

Article Title: Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

doi: 10.1530/rep-18-0333

Figure Lengend Snippet: Figure 3 Cyclic changes in versican expression in human endometrium. (A) Lysates of isolated EECs were subjected to western blotting using the same anti-versican antibody as in Fig. 2A, which is specific for versican V0 and V1. The lower panel shows GAPDH as control. Versican V1 was detected as multiple protein signals ranging from 440 to 500 kDa. (B) Protein signal intensities for versican V1 were significantly higher in secretory phases than in proliferative phases. Error bars represent standard deviation. *P < 0.05.

Article Snippet: Goat anti-human versican polyclonal antibody (pAb; #AF3054), which recognizes an amino acid sequence in the GAG-β domain specific to the V0 and V1 isoforms, was purchased from R&D Systems.

Techniques: Expressing, Isolation, Western Blot, Control, Standard Deviation

Journal: Reproduction

Article Title: Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

doi: 10.1530/rep-18-0333

Figure Lengend Snippet: Figure 4 Effects of ovarian steroid hormones on versican expression in isolated EECs. (A) EECs were isolated from the endometrial tissue of cycle day 12 and cultured for 24 h (day 1) or 72 h (day 3). Note the intense versican deposition in the extracellular space on day 3, indicating that versican secretion from EECs increased in a time-dependent manner. (a, c) and (b, d) represent immunofluorescence and phase-contrast images, respectively. Scale bars = 100 µm. (B) Isolated EECs were treated with ethanol (EtOH), 10 nM estradiol (E2) or 10 nM E2 plus 1 µM progesterone (P4) and then immunostained. The final concentration of EtOH was 0.1% (v/v). Versican expression levels in EECs were measured by mean fluorescent intensity. The sum of the integrated densities from five areas (central, upper, lower, right and left) in the immunostained specimen is defined as the ‘versican expression level’. The versican expression level was significantly higher after treatment with E2 and P4 (E2 + P4) than after treatment with EtOH or E2 alone (n = 5). Error bars represent standard deviation. *P < 0.05.

Article Snippet: Goat anti-human versican polyclonal antibody (pAb; #AF3054), which recognizes an amino acid sequence in the GAG-β domain specific to the V0 and V1 isoforms, was purchased from R&D Systems.

Techniques: Expressing, Isolation, Cell Culture, Immunofluorescence, Concentration Assay, Standard Deviation

Journal: Reproduction

Article Title: Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

doi: 10.1530/rep-18-0333

Figure Lengend Snippet: Figure 6 Differential effects of versican V1 and V3 on BeWo spheroid attachment. (A) Phase-contrast images of BeWo spheroids after 1-h incubation on a monolayer of (a) GFP-overexpressing Ishikawa cells (ISKW-GFP), (b) versican V1-overexpressing Ishikawa cells (ISKW-V1) and (c) versican V3-overexpressing Ishikawa cells (ISKW-V3). Note that all spheroids were attached to the (b) ISKW-V1 monolayer, whereas some spheroids were floating (arrowheads) over the (a) ISKW-GFP and (c) ISKW-V3 monolayers. Scale bars = 200 µm. (B) Attachment ratios to the ISKW-V1 monolayer (n = 4) were significantly higher compared with that of the control ISKW-GFP monolayer (n = 4). (C) Attachment ratios to the ISKW-V3 monolayer (n = 6) were not significantly different than those of the control (n = 6). All values are relative to the control, ISKW-GFP. In all panels, error bars represent standard deviation. *P < 0.05; N.S., not significant.

Article Snippet: Goat anti-human versican polyclonal antibody (pAb; #AF3054), which recognizes an amino acid sequence in the GAG-β domain specific to the V0 and V1 isoforms, was purchased from R&D Systems.

Techniques: Incubation, Control, Standard Deviation

Journal: Reproduction

Article Title: Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

doi: 10.1530/rep-18-0333

Figure Lengend Snippet: Figure 5 Characterization of versican V1- and V3-overexpressing Ishikawa cells. (A) Cell lysates extracted from original Ishikawa cells (Ishikawa; lane 1), platform Ishikawa cells (ISKW; lane 2), GFP- overexpressing Ishikawa cells (ISKW-GFP; lane 3) and versican V1-overexpressing Ishikawa cells (ISKW-V1; lane 4) were subjected to western blotting. ISKW possesses a specific sequence into which foreign DNA can be inserted by a recombinase. An intense protein signal at 440 kDa corresponding to versican V1 is only detectable in ISKW-V1. (B) Cell lysates from ISKW-V1 (lane 1), conditioned medium derived from ISKW-V1 (V1-CM; lane 2) and conditioned medium derived from ISKW-GFP (GFP-CM; lane 3) were subjected to western blotting using the same anti-versican antibody as in (A). Note that the protein signal at 440kDa is detected in both ISKW-V1 cell lysates (lane 1) and V1-CM (lane 2). (C) Cell lysates extracted from original Ishikawa cells (Ishikawa; lane 1), platform Ishikawa cells (ISKW; lane 2), GFP-overexpressing Ishikawa cells (ISKW-GFP; lane 3), and versican V3-overexpressing Ishikawa cells (ISKW-V3; lane 4) were subjected to western blotting using an anti-versican monoclonal antibody raised against the G3 domain common in all versican isoforms. An intense protein signal at 76 kDa corresponding to versican V3 was only detected in ISKW-V3. (D) Cell lysates from ISKW-V3 (lane1), conditioned medium derived from ISKW-V3 (V3-CM; lane2) and CM derived from ISKW-GFP (GFP-CM; lane 3) were subjected to western blotting using the same anti-versican antibody as in (C). Note that the protein signal at 76 kDa is only detectable in the ISKW-V3 cell lysates (lane 1) but not in V3-CM (lane 2). (E) ISKW-V1 cells cultured for 48 h were subjected to immunocytochemistry using the same anti-versican antibody as in (A). Note that intense immunoreactive versican is detected not only on the cell surface but also in the extracellular space (circumscribed with the dotted line). Images (a) and (b) represent immunofluorescence and phase-contrast images, respectively. Scale bars = 100 µm. (F) ISKW-V3 cells cultured for 48 h were subjected to immunocytochemistry using anti-versican polyclonal antibody raised against the G1 domain common in all versican isoforms. Note that intense immunoreactive versican is detected exclusively on the cell surface and not in the extracellular space (circumscribed with the dotted line). Images (a) and (b) represent immunofluorescence and phase-contrast images, respectively. Scale bars = 100 µm.

Article Snippet: Goat anti-human versican polyclonal antibody (pAb; #AF3054), which recognizes an amino acid sequence in the GAG-β domain specific to the V0 and V1 isoforms, was purchased from R&D Systems.

Techniques: Western Blot, Sequencing, Derivative Assay, Cell Culture, Immunocytochemistry, Immunofluorescence

Journal: Reproduction

Article Title: Versican V1 in human endometrial epithelial cells promotes BeWo spheroid adhesion in vitro

doi: 10.1530/rep-18-0333

Figure Lengend Snippet: Figure 7 Versican V1 promotion of BeWo spheroid attachment requires the presence of chondroitin sulfate side chains. (A) BeWo spheroids are incubated on the monolayer of platform Ishikawa cells (ISKW) in the presence of conditioned medium derived from GFP-overexpressing Ishikawa cells (GFP-CM) or that derived from versican V1-overexpressing Ishikawa cells (V1-CM). The attachment ratio is significantly higher in the presence of V1-CM (n = 6) than in the presence of GFP-CM (n = 6). All values are relative to the control, GFP-CM. (B) BeWo spheroids were incubated on ISKW monolayers in the presence of GFP-CM or V1-CM with or without chondroitinase ABC (ChABC) pretreatment. The attachment ratio significantly increased in the presence of V1-CM (n = 7) compared with that of GFP-CM (n = 7). Note that the attachment- promoting effect of V1-CM was completely abrogated by ChABC pretreatment (n = 7). All values are relative to the control, GFP-CM, without ChABC treatment. (C) BeWo spheroids were incubated on ISKW monolayers in the presence of V1-CM with or without hyaluronidase (Hase) pretreatment. Note that Hase pretreatment did not affect the attachment ratio that was increased by untreated V1-CM (n = 4). In all panels, error bars represent standard deviation. *P < 0.05; N.S., not significant.

Article Snippet: Goat anti-human versican polyclonal antibody (pAb; #AF3054), which recognizes an amino acid sequence in the GAG-β domain specific to the V0 and V1 isoforms, was purchased from R&D Systems.

Techniques: Incubation, Derivative Assay, Control, Standard Deviation