Journal: Upsala Journal of Medical Sciences

Article Title: Surface glycans contribute to differences between seminal prostasomes from normozoospermic and oligozoospermic men

doi: 10.1080/03009734.2019.1592266

Figure Lengend Snippet: Surface glycosylation of seminal prostasomes: lectin- and immune-transmission electron microscopy. A: Lectin-TEM using SNA. Inserts show enlarged characteristic pattern of SNA-reactivity to each sample group. B: Lectin-TEM using ConA. Inserts show enlarged characteristic pattern of ConA-reactivity to vesicles in each sample group. In O, staining of some proteinaceous material was also observed (arrowheads). C: Immune-TEM using anti-galectin-3 antibodies. Inserts show enlarged characteristic pattern of anti-gal-3-reactivity to each sample group. Micrographs show most characteristic patterns obtained. Although differences in the reactivity of particular vesicles could be noticed, it does not affect the general reactivity of the sample (as in IEC when taking all vesicles into account). (N = seminal prostasomes from normozoospermic men; O = seminal prostasomes from oligozoospermic men).

Article Snippet: Monoclonal anti-CD63 antibody (clone TS63) was from Abcam (Cambridge, UK) and biotinylated goat anti-galectin-3 (gal-3) antibodies from R&D Systems (Minneapolis, MN, USA); 3,3′,5,5′-tetramethylbenzidine (TMB), bovine serum albumin (BSA), D-lactose, and methyl- alpha D-mannopyranoside were from Sigma (St. Louis, MO, USA).

Techniques: Transmission Assay, Electron Microscopy, Staining

Journal: Biomolecules

Article Title: Exploration into Galectin-3 Driven Endocytosis and Lattices

doi: 10.3390/biom14091169

Figure Lengend Snippet: Gal3-based duality in β 1 integrin dynamics. ( A ) Schematic representation of the molecular organization of Gal3 where the C-terminal carbohydrate recognition domain (CRD) and the N-terminal oligomerization domain are indicated. ( B ) Left: Representative region of interest (leading edge) of a 2D STORM image of an RPE-1 cell showing surface-bound Gal3 ( top ) and the corresponding clusters obtained after segmentation ( bottom ). Right : The occurrence of each type of Gal3 cluster is shown in function of their surface area (×10 3 nm 2 ). Means ± SEM, one-way ANOVA; ns = p > 0.05, **** p < 0.0001. Scale bar = 5 μm. ( C ) The percentage of Gal3 molecules is shown in function of the different Gal3 cluster populations. Means ± SEM, one-way ANOVA; ns = p > 0.05, * p < 0.05, ** p < 0.002, **** p < 0.0001. ( D ) 2D STORM image of Gal3 and anti-β 1 integrin antibodies on RPE-1 cells (4 °C co-binding). A zoom of the leading edge is shown to illustrate the extensive level of colocalization between Gal3 and β 1 integrin. Gal3 clusters with variable size and shape are detected (yellow arrowheads in the zoom). Scale bars = 5 μm. (E) Quantification of the probability of proximity (colocalization) between Gal3 and β 1 integrin for each class of Gal3 clusters, compared to random non-clustered Gal3. Means ± SEM, one-way ANOVA; ns = p > 0.05, **** p < 0.0001. ( F ) Top: Anti-β 1 integrin antibody uptake assay in RPE-1 cells. Internalized antibody is immunolabeled, imaged by confocal microscopy, and quantified. Bottom: Scheme of the protocol detailing the use of the Gal3 inhibitor I3 (10 μM) in acute versus prolonged incubation conditions. ( G ) Anti-β 1 integrin antibody uptake assay as in ( F ). Note the shift from both peripheral (green arrowheads) and perinuclear (red arrowheads) distribution of internalized β 1 integrin in control cells to exclusive perinuclear localization in the prolonged incubation condition. ( H ) Transferrin (Tf) internalization is very little affected by I3. In ( G ) and ( H ): Dashed lines indicate the contour of individual cells. Scale bars = 10 μm. Nuclei in blue (DAPI). Quantification of fluorescence intensities as means ± SEM, one-way ANOVA; ns = p > 0.05, ** p < 0.002, **** p < 0.0001.

Article Snippet: Reagents: Anti-β 1 mAb13 antibody (BD Bioscience, Le Pont de Claix, France; Ref. 552828), anti-Gal3 antibody (Fu-Tong Liu, UC Davis, CA, USA), Cy3-labeled anti-β 1 integrin mAb13 antibody, anti-EEA1 antibody (Biorbyt, Durham, US; Ref. 11606), Cy3-labeled donkey anti-rat antibody (Beckman Coulter, Roissy CDG, France; Ref. 712-166-153), Cy3-labeled anti-goat antibody, fluorescein-isothiocyanate dextran 70 kDa (Sigma Merck, Saint Quentin Fallavier, France; Ref. 46945), Gal3 inhibitor compound I3 (1,1′-sulfanediyl-bis-{3-deoxy-3-[4-(butylaminocarbonyl)-1H-1,2,3-triazol-1-yl]-β-D galactopyranoside} [ , ], Genz-123346 (Sigma Merck, Saint Quentin Fallavier, France; Ref. 5382850001), β-D-lactose (Sigma Merck, Saint Quentin Fallavier, France; Ref. L3750), NHS-Cy3 (GE, Buc, France; PA23001), NHS-CF680 (Sigma Merck, Saint Quentin Fallavier, France; SCJ4600055), transferrin-Alexa546 (Tf-A546) (Invitrogen, Saint Aubin, France; Ref. T23364), AlexaFluor488-labeled recombinant purified Gal3, Cy3-labeled recombinant purified Gal3, AlexaFluor647-labeled recombinant purified Gal3.

Techniques: Binding Assay, Immunolabeling, Confocal Microscopy, Incubation, Control, Fluorescence

Journal: Biomolecules

Article Title: Exploration into Galectin-3 Driven Endocytosis and Lattices

doi: 10.3390/biom14091169

Figure Lengend Snippet: GSL-based duality in β 1 integrin dynamics. ( A ) Simplified schematic representation of the early steps of GSL synthesis. The reaction inhibited by Genz-123346 is indicated. ( B ) Analysis of cellular levels of the indicated GSL in function of incubation time with Genz-123346. Note that the most important drop occurs up to day 3. Means ± SEM, unpaired t -test; ns = p > 0.05, ** p < 0.002, **** p < 0.0001. ( C ) Scheme of experimental procedure detailing how GSL inhibition has been set up either in acute (3 days) or prolonged (5 days) incubation conditions, prior to cargo protein internalization for 10 min. ( D ) Anti-β 1 integrin antibody uptake assay as in ( C ). Note that β 1 integrin uptake is inhibited upon acute Genz-123346 treatment and increased upon prolonged treatment. In the latter condition, the intracellular accumulation of β 1 integrin is massively perinuclear (red arrowheads), compared to control cells where peripheral localizations are also observed (green arrowheads). Means ± SEM, unpaired t -test; **** p < 0.0001. ( E ) Transferrin (Tf) internalization (10 min) is only mildly affected in all conditions. Means ± SEM, unpaired t -test; ns = p > 0.05, ** p < 0.002. ( F ) Internalization of exogenous Gal3 (10 min). Similar to β 1 integrin, Gal3 endocytosis is significantly inhibited upon acute Genz-123346 treatment, and increased with perinuclear accumulation upon prolonged treatment (red arrowheads). Means ± SEM, unpaired t -test; **** p < 0.0001. In ( D – F ): Yellow dashed lines indicate contours of cells; scale bars = 10 μm, nuclei in blue (DAPI).

Article Snippet: Reagents: Anti-β 1 mAb13 antibody (BD Bioscience, Le Pont de Claix, France; Ref. 552828), anti-Gal3 antibody (Fu-Tong Liu, UC Davis, CA, USA), Cy3-labeled anti-β 1 integrin mAb13 antibody, anti-EEA1 antibody (Biorbyt, Durham, US; Ref. 11606), Cy3-labeled donkey anti-rat antibody (Beckman Coulter, Roissy CDG, France; Ref. 712-166-153), Cy3-labeled anti-goat antibody, fluorescein-isothiocyanate dextran 70 kDa (Sigma Merck, Saint Quentin Fallavier, France; Ref. 46945), Gal3 inhibitor compound I3 (1,1′-sulfanediyl-bis-{3-deoxy-3-[4-(butylaminocarbonyl)-1H-1,2,3-triazol-1-yl]-β-D galactopyranoside} [ , ], Genz-123346 (Sigma Merck, Saint Quentin Fallavier, France; Ref. 5382850001), β-D-lactose (Sigma Merck, Saint Quentin Fallavier, France; Ref. L3750), NHS-Cy3 (GE, Buc, France; PA23001), NHS-CF680 (Sigma Merck, Saint Quentin Fallavier, France; SCJ4600055), transferrin-Alexa546 (Tf-A546) (Invitrogen, Saint Aubin, France; Ref. T23364), AlexaFluor488-labeled recombinant purified Gal3, Cy3-labeled recombinant purified Gal3, AlexaFluor647-labeled recombinant purified Gal3.

Techniques: Incubation, Inhibition, Control

Journal: Biomolecules

Article Title: Exploration into Galectin-3 Driven Endocytosis and Lattices

doi: 10.3390/biom14091169

Figure Lengend Snippet: Characterization of sites of perinuclear β 1 integrin accumulation. ( A , B ) Anti-β 1 integrin or ( C ) Gal3 uptake assay (10 min) under acute or prolonged I3 ( A ) or Genz-123346 ( B , C ) treatment followed by immunolabeling for EEA1. The colocalization of β 1 integrin ( A , B ) or Gal3 ( C ) with EEA1 as well as the fluorescent intensity of EEA1 signal were quantified ( right ). Note the increased colocalization of internalized β 1 integrin ( A , B ) or Gal3 ( C ) with EEA1 and increased EEA1 signal intensity, notably in the prolonged treatment conditions. Means ± SEM, one-way ANOVA ( A , B ), or unpaired t -test ( C ); **** p < 0.0001. Yellow dashed lines indicate contours of cells. Scale bars = 10 μm, nuclei in blue (DAPI).

Article Snippet: Reagents: Anti-β 1 mAb13 antibody (BD Bioscience, Le Pont de Claix, France; Ref. 552828), anti-Gal3 antibody (Fu-Tong Liu, UC Davis, CA, USA), Cy3-labeled anti-β 1 integrin mAb13 antibody, anti-EEA1 antibody (Biorbyt, Durham, US; Ref. 11606), Cy3-labeled donkey anti-rat antibody (Beckman Coulter, Roissy CDG, France; Ref. 712-166-153), Cy3-labeled anti-goat antibody, fluorescein-isothiocyanate dextran 70 kDa (Sigma Merck, Saint Quentin Fallavier, France; Ref. 46945), Gal3 inhibitor compound I3 (1,1′-sulfanediyl-bis-{3-deoxy-3-[4-(butylaminocarbonyl)-1H-1,2,3-triazol-1-yl]-β-D galactopyranoside} [ , ], Genz-123346 (Sigma Merck, Saint Quentin Fallavier, France; Ref. 5382850001), β-D-lactose (Sigma Merck, Saint Quentin Fallavier, France; Ref. L3750), NHS-Cy3 (GE, Buc, France; PA23001), NHS-CF680 (Sigma Merck, Saint Quentin Fallavier, France; SCJ4600055), transferrin-Alexa546 (Tf-A546) (Invitrogen, Saint Aubin, France; Ref. T23364), AlexaFluor488-labeled recombinant purified Gal3, Cy3-labeled recombinant purified Gal3, AlexaFluor647-labeled recombinant purified Gal3.

Techniques: Immunolabeling

Journal: Biomolecules

Article Title: Exploration into Galectin-3 Driven Endocytosis and Lattices

doi: 10.3390/biom14091169

Figure Lengend Snippet: Exogenous Gal3 and dextran 70K uptake upon prolonged GSL depletion. After prolonged (5 days) treatment with Genz-123346, RPE-1 cells were continuously co-incubated (10 min) with exogenous Gal3 and dextran 70K. Note the increased perinuclear accumulation of Gal3 and its increased overlap with dextran 70K under these conditions. Means ± SEM, unpaired t -test; **** p < 0.0001. Yellow dashed lines indicate contours of cells. Scale bars = 10 μm, nuclei in blue (DAPI).

Article Snippet: Reagents: Anti-β 1 mAb13 antibody (BD Bioscience, Le Pont de Claix, France; Ref. 552828), anti-Gal3 antibody (Fu-Tong Liu, UC Davis, CA, USA), Cy3-labeled anti-β 1 integrin mAb13 antibody, anti-EEA1 antibody (Biorbyt, Durham, US; Ref. 11606), Cy3-labeled donkey anti-rat antibody (Beckman Coulter, Roissy CDG, France; Ref. 712-166-153), Cy3-labeled anti-goat antibody, fluorescein-isothiocyanate dextran 70 kDa (Sigma Merck, Saint Quentin Fallavier, France; Ref. 46945), Gal3 inhibitor compound I3 (1,1′-sulfanediyl-bis-{3-deoxy-3-[4-(butylaminocarbonyl)-1H-1,2,3-triazol-1-yl]-β-D galactopyranoside} [ , ], Genz-123346 (Sigma Merck, Saint Quentin Fallavier, France; Ref. 5382850001), β-D-lactose (Sigma Merck, Saint Quentin Fallavier, France; Ref. L3750), NHS-Cy3 (GE, Buc, France; PA23001), NHS-CF680 (Sigma Merck, Saint Quentin Fallavier, France; SCJ4600055), transferrin-Alexa546 (Tf-A546) (Invitrogen, Saint Aubin, France; Ref. T23364), AlexaFluor488-labeled recombinant purified Gal3, Cy3-labeled recombinant purified Gal3, AlexaFluor647-labeled recombinant purified Gal3.

Techniques: Incubation

Journal: Biomolecules

Article Title: Exploration into Galectin-3 Driven Endocytosis and Lattices

doi: 10.3390/biom14091169

Figure Lengend Snippet: Role of clathrin in endocytic uptake under prolonged treatment conditions. ( A – C ) Uptake assays (10 min) of anti-β 1 integrin antibodies ( A , B ) or Gal3 ( C ) upon prolonged I3 ( A ) or Genz-123346 ( B , C ) treatment. When indicated (siCHC), clathrin heavy chain was depleted ( right images). The perinuclear accumulation of β 1 integrin ( A , B ) or that of Gal3 ( C ) as observed in the prolonged treatment conditions (red or white arrowheads) is strongly inhibited upon clathrin depletion. Means ± SEM, one-way ANOVA; **** p < 0.0001. Yellow dashed lines indicate contours of cells. Scale bars = 10 μm, nuclei in blue (DAPI).

Article Snippet: Reagents: Anti-β 1 mAb13 antibody (BD Bioscience, Le Pont de Claix, France; Ref. 552828), anti-Gal3 antibody (Fu-Tong Liu, UC Davis, CA, USA), Cy3-labeled anti-β 1 integrin mAb13 antibody, anti-EEA1 antibody (Biorbyt, Durham, US; Ref. 11606), Cy3-labeled donkey anti-rat antibody (Beckman Coulter, Roissy CDG, France; Ref. 712-166-153), Cy3-labeled anti-goat antibody, fluorescein-isothiocyanate dextran 70 kDa (Sigma Merck, Saint Quentin Fallavier, France; Ref. 46945), Gal3 inhibitor compound I3 (1,1′-sulfanediyl-bis-{3-deoxy-3-[4-(butylaminocarbonyl)-1H-1,2,3-triazol-1-yl]-β-D galactopyranoside} [ , ], Genz-123346 (Sigma Merck, Saint Quentin Fallavier, France; Ref. 5382850001), β-D-lactose (Sigma Merck, Saint Quentin Fallavier, France; Ref. L3750), NHS-Cy3 (GE, Buc, France; PA23001), NHS-CF680 (Sigma Merck, Saint Quentin Fallavier, France; SCJ4600055), transferrin-Alexa546 (Tf-A546) (Invitrogen, Saint Aubin, France; Ref. T23364), AlexaFluor488-labeled recombinant purified Gal3, Cy3-labeled recombinant purified Gal3, AlexaFluor647-labeled recombinant purified Gal3.

Techniques:

Journal: Biomolecules

Article Title: Exploration into Galectin-3 Driven Endocytosis and Lattices

doi: 10.3390/biom14091169

Figure Lengend Snippet: Continuum model between lattices and GL-Lect driven endocytosis. ( A ) Unperturbed condition. A glycoprotein cargo, here α 5 β 1 integrin, is either recruited into galectin lattices ( left , underlined in red) or internalized by GL-Lect driven endocytosis ( right , underlined in blue). ( B ) Acute treatment conditions. Since tubular endocytic pits for GL-Lect driven endocytosis are built de novo, acute interference with Gal3 activity or GSL expression prevents their formation. In contrast, preassembled galectin lattices resist under these conditions. ( C ) Prolonged treatment conditions. Even galectin lattices are disassembled. With GL-Lect driven endocytosis being inhibited, α 5 β 1 integrin is now internalized by alternative endocytic pathways, i.e., clathrin-mediated endocytosis and macropinocytosis.

Article Snippet: Reagents: Anti-β 1 mAb13 antibody (BD Bioscience, Le Pont de Claix, France; Ref. 552828), anti-Gal3 antibody (Fu-Tong Liu, UC Davis, CA, USA), Cy3-labeled anti-β 1 integrin mAb13 antibody, anti-EEA1 antibody (Biorbyt, Durham, US; Ref. 11606), Cy3-labeled donkey anti-rat antibody (Beckman Coulter, Roissy CDG, France; Ref. 712-166-153), Cy3-labeled anti-goat antibody, fluorescein-isothiocyanate dextran 70 kDa (Sigma Merck, Saint Quentin Fallavier, France; Ref. 46945), Gal3 inhibitor compound I3 (1,1′-sulfanediyl-bis-{3-deoxy-3-[4-(butylaminocarbonyl)-1H-1,2,3-triazol-1-yl]-β-D galactopyranoside} [ , ], Genz-123346 (Sigma Merck, Saint Quentin Fallavier, France; Ref. 5382850001), β-D-lactose (Sigma Merck, Saint Quentin Fallavier, France; Ref. L3750), NHS-Cy3 (GE, Buc, France; PA23001), NHS-CF680 (Sigma Merck, Saint Quentin Fallavier, France; SCJ4600055), transferrin-Alexa546 (Tf-A546) (Invitrogen, Saint Aubin, France; Ref. T23364), AlexaFluor488-labeled recombinant purified Gal3, Cy3-labeled recombinant purified Gal3, AlexaFluor647-labeled recombinant purified Gal3.

Techniques: Activity Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Prototype and Chimera-Type Galectins in Placentas with Spontaneous and Recurrent Miscarriages

doi: 10.3390/ijms17050644

Figure Lengend Snippet: Antibodies used in study for immunohistochemistry.

Article Snippet: Gal-3 , 9C4 , mouse , 4.6 mg/mL 1:1000 in PBS , Novocastra, Wetzlar, Germany , Vectastain Elite Kit (Linaris) , Colon.

Techniques: Immunohistochemistry, Concentration Assay, Positive Control, Blocking Assay