Gal-4 mRNA expression in pancreatic cancer cell lines.

<p>Gal-4 mRNA expression of normal human pancreatic duct epithelial-like cell line (hTERT-HPNE) and 9 different human pancreatic cancer cell lines was analyzed by quantitative real-time PCR and depicted as the relative amount of Gal-4 transcripts (± SEM) compared to the expression of the endogenous reference gene <i>GAPDH</i>. (*** p≤0.001 versus all other cell lines, using one way ANOVA with post Dunnett two sided t tests).</p

In vitro cell migration of PaTu-T cells.

<p>A scratch (wound healing) assay was performed with PaTu-T, PaTu-T/Gal-4 and PaTu-T/mock cells. PaTu-T/mock and PaTu-T/Gal-4 cells were seeded on a 24 well plate and scratched on the surface with a 200-µl pipette tip. Relative values were set at 100% of the gap width at the time of the scratch. <b>A)</b> Representative photographs at time points 0, 6, 19 and 24 hours after the wound (scratch) for all conditions are depicted. <b>B)</b> Histogram representation of data analyzed from photographs taken at 0 h; 6 h, 19 h and 24 h after the scratch. Measurements were done in duplicate in 3 separate experiments, and data are depicted as average gap width ± SEM. (* p≤0.05 and ** p≤0.01, using one way ANOVA Tukey t tests).</p

Gal-4 and Gal-4 binding sites in PaTu-S and PaTu-T cells.

<p>Detection of endogenous Gal-4, and Gal-4 ligands, in PaTu-S and PaTu-T cells by flow cytometry. A histogram of one representative experiment is depicted for each condition of least two independent experiments. <b>A)</b> Dot plots of Gal-4 staining of permeabilized PaTu-S and PaTu-T cells. Gal-4 was detected at 4°C with anti-hGal-4 Abs in fixed permeabilized cells. Secondary Abs staining without anti-hGal-4 Abs was used as background autofluorescence control. <b>B)</b> Presence of endogenous bound Gal-4 to the surface of PaTu-S and PaTu-T after washing the cells with 500 mM lactose prior to Gal-4 staining. The presence of Gal-4 was established by FACS analysis using anti-hGal-4 Abs at 4°C. Endogenous Gal-4 bound to the surface is shown by a black line. <b>C)</b> The presence of Gal-4 binding sites on PaTu-S and PaTu-T cells was determined after washing the cells with 500 mM lactose prior to Gal-4 staining. The binding of externally added recombinant (rec) hGal-4 (5 µg/ml, black line) was investigated. Binding of rec hGal-4 to the surface could be inhibited by adding lactose (dark field). Background staining with secondary Abs is depicted as light grey fields in B and C.</p

Immunocytochemical localization of Gal-4 in PaTu-S cells.

<p>Photographs of representative ICC analysis of the cellular localization of Gal-4 in PaTu-S cells. Gal-4 was detected using Alexa-labeled anti-Gal-4 Abs (green), Actin was stained using Phalloidin (red) and nucleus staining obtained using HOESCHS (blue); the third panel shows the merging of the different stainings. Bar = 25 µm.</p

High Sensitive Detection of Carbohydrate Binding Proteins in an ELISA-Solid Phase Assay Based on Multivalent Glyconanoparticles

<div><p>Improved detection of anti-carbohydrate antibodies is a need in clinical identification of biomarkers for cancer cells or pathogens. Here, we report a new ELISA approach for the detection of specific immunoglobulins (IgGs) against carbohydrates. Two nanometer gold glyconanoparticles bearing oligosaccharide epitopes of HIV or <i>Streptococcus pneumoniae</i> were used as antigens to coat ELISA-plates. A ~3,000-fold improved detection of specific IgGs in mice immunized against <i>S. pneumoniae</i> respect to the well known BSA-glycoconjugate ELISA was achieved. Moreover, these multivalent glyconanoparticles have been employed in solid phase assays to detect the carbohydrate-dependent binding of human dendritic cells and the lectin DC-SIGN. Multivalent glyconanoparticles in ELISA provide a versatile, easy and highly sensitive method to detect and quantify the binding of glycan to proteins and to facilitate the identification of biomarkers.</p> </div

GNP-ELISA for the detection of anti-carbohydrates antibodies from mice immunized with TetraPnOv-GNP.

<p>(<b>A</b>) Detection of specific IgG by GNPs carrying different carbohydrates. TetraPnOv- and TetraPn-GNPs show strong binding to mice serum at a 1:30,000 dilution. Detectable binding was also observed for Gal-GNP. Glc-, TetraMan-, and DiMan-GNPs were not recognized by the sera’s IgG. Non-specific interactions of the secondary anti-mouse IgG with the GNPs were excluded performing the GNP-ELISA in the absence of 2G12. Sera of mice immunized with saline were used as negative control. Differences between sera from immunized mice and control samples are significant, as indicated with one (<i>p</i><0.05) or two asterisks (<i>p</i><0.01); (<b>B</b>) ELISA plate coated with 25 µg/mL of TetraPn-GNP carrying <i>S</i>. <i>pneumoniae</i> or Glc-GNP (control) were used to determine the detection limit for anti-TetraPn antibodies in mice sera. GNP-ELISA was able to detect antibodies up to 1:50,000 dilutions of sera. Differences between TetraMan-GNP and Glc- GNP are significant, as indicated with one (<i>p</i><0.05) or two asterisks (<i>p</i><0.01).</p

DC-SIGN-Fc binding to GNPs.

<p>Binding was determined using GNPs-ELISA in PBS and in calcium and magnesium containing buffer (TMS). These experiments were performed in triplicate at least three times with similar results. Error bars indicate standard deviations.</p

Oligonucleotide primers for quantitative RT-PCR.

<p>^{SOCS, Suppressor of Cytokine Signalling; SHP, SH2-containing protein tyrosine Phosphatase; PD-L1, Programmed Death Ligand 1;TGF, Transforming Growth Factor; GAPDH, Glyceraldehyde 3-Phosphatase Dehydrogenase}</p><p>Oligonucleotide primers for quantitative RT-PCR.</p

Dendritic cells adhesion assay using GNP-ELISA.

<p>Dendritic cells show different carbohydrate-affinity. Binding of moDC to GNPs in calcium and magnesium containing buffer, was determined using plate adhesion assay in the presence or absence of EGTA (3.75 nM) or anti-DC-SIGN antibody AZN-D1 (10 μg/mL). These experiments were performed at least four times with similar results. Each experiment was performed in triplicate. Error bars indicate standard deviations. The binding to the GNPs was significantly decreased when treated with EGTA or AZN-D1, as indicated with one (<i>p</i><0.05) or two asterisks (<i>p</i><0.01).</p

Direct comparison between BSA-ELISA and GNP-ELISA for IgGs detection in mice.

<p>At low sera dilution (dotted blue lines, from 1:10 to 1:200) the BSA-ELISA (blue line, squares) gave a response from 0.7 to 0.1 OD, while the GNP-ELISA (blue line, triangle) gave a saturated response around 4 OD. At high sera dilution (dark lines, from 1:10.000 to 1:100.000) the BSA-ELISA (dark line, reverse triangle) was no able to detect IgGs (OD around zero), while the GNP-ELISA (dark line, rhombus) detected IgGs (OD from 1.4 to 0.3) up to 1:50.000 diluted sera.</p