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: Stem Cell Research & Therapy

Article Title: Galectin-1 and galectin-3 expression in equine mesenchymal stromal cells (MSCs), synovial fibroblasts and chondrocytes, and the effect of inflammation on MSC motility

doi: 10.1186/s13287-017-0691-2

Figure Lengend Snippet: Constitutive galectin gene expression. Galectin-1 and galectin-3 transcription levels (copy number/ng RNA) from ( a ) equine BMSCs ( MSC , n = 31), cultured synoviocytes ( Syn , n = 27) and freshly harvested carpal synovial membrane tissue ( Syn Memb , n = 23) and ( b ) equine BMSCs ( MSC , n = 31), cultured chondrocytes ( Chond , n = 16) and freshly harvested carpal articular cartilage ( Cart , n = 10). Data are presented as mean ± SE. Statistical analysis is performed on log-transformed data (** p < 0.01, *** p < 0.001, **** p < 0.0001, Tukey’s post hoc tests)

Article Snippet: 10 μL of sample per well was loaded onto a 7.5% TGX gel (Bio-Rad, Hercules, CA, USA), and subjected to SDS-PAGE for 1 h at 100 V. Gels were transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA), and immunoblotting was performed using antibodies against galectin-1 (R&D Systems, Minneapolis, MN, USA) goat anti-mouse Gal-1 pAb, AF1245), galectin-3 (Santa Cruz Biotechnology, Dallas, TX, USA; goat anti-human Gal-3 pAb, sc-19280) and actin (Santa Cruz Biotechnology, Dallas, TX, USA; goat anti-human actin pAb, sc-1615).

Techniques: Expressing, Cell Culture, Transformation Assay

Journal: Stem Cell Research & Therapy

Article Title: Galectin-1 and galectin-3 expression in equine mesenchymal stromal cells (MSCs), synovial fibroblasts and chondrocytes, and the effect of inflammation on MSC motility

doi: 10.1186/s13287-017-0691-2

Figure Lengend Snippet: Inflammation decreases galectin gene expression in BMSCs. Galectin-1 and galectin-3 expression levels are reduced following exposure to pro-inflammatory stimuli. a Galectin-1 and ( b ) galectin-3 transcription levels (copy number/ng RNA) in equine passage 3 BMSCs 20 h after stimulation with recombinant equine interleukin-1 beta ( IL-1β ), recombinant equine tumor necrosis factor alpha ( TNF-α ), or lipopolysaccharide ( LPS ). Data are presented as mean ± SE of three independent experiments in which three different BMSC donors were analyzed in duplicate. Statistical analysis is performed using Friedman’s test with post hoc comparisons as described by Gibbons and Chakraborti (p. 459, equation 2.13) using a Bonferroni correction for multiple comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, ****p < 0.0001)

Article Snippet: 10 μL of sample per well was loaded onto a 7.5% TGX gel (Bio-Rad, Hercules, CA, USA), and subjected to SDS-PAGE for 1 h at 100 V. Gels were transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA), and immunoblotting was performed using antibodies against galectin-1 (R&D Systems, Minneapolis, MN, USA) goat anti-mouse Gal-1 pAb, AF1245), galectin-3 (Santa Cruz Biotechnology, Dallas, TX, USA; goat anti-human Gal-3 pAb, sc-19280) and actin (Santa Cruz Biotechnology, Dallas, TX, USA; goat anti-human actin pAb, sc-1615).

Techniques: Expressing, Recombinant

Journal: Stem Cell Research & Therapy

Article Title: Galectin-1 and galectin-3 expression in equine mesenchymal stromal cells (MSCs), synovial fibroblasts and chondrocytes, and the effect of inflammation on MSC motility

doi: 10.1186/s13287-017-0691-2

Figure Lengend Snippet: Secreted and intracellular/membrane-bound galectins in BMSCs. a Galectin-1 and ( b ) galectin-3 secretion (μg/mL) measured in the supernatants of cytokine- or LPS-treated equine passage 3 BMSCs 20 h following treatment. c Galectin-1 and ( d ) galectin-3 concentrations measured in cell lysates from cytokine- or lipopolysaccharide ( LPS )-treated equine passage 3 BMSCs 20 h following treatment. For all graphs, data are presented as mean ± SE of three independent experiments in which three different BMSC donors were analyzed in duplicate. Statistical analysis is performed using Friedman’s test with post hoc comparisons as described by Gibbons and Chakraborti (p. 459, equation 2.13) using a Bonferroni correction for multiple comparisons. Galectin-1 concentrations were increased only in supernatants from MSCs exposed to the 50 μg/mL dose of LPS (** p < 0.01). No significant differences were observed for galectin-3 supernatants or galectin-1/galectin-3 cell lysates. IL-1β interleukin-1 beta; TNF-α tumor necrosis factor alpha

Article Snippet: 10 μL of sample per well was loaded onto a 7.5% TGX gel (Bio-Rad, Hercules, CA, USA), and subjected to SDS-PAGE for 1 h at 100 V. Gels were transferred to PVDF membranes (EMD Millipore, Billerica, MA, USA), and immunoblotting was performed using antibodies against galectin-1 (R&D Systems, Minneapolis, MN, USA) goat anti-mouse Gal-1 pAb, AF1245), galectin-3 (Santa Cruz Biotechnology, Dallas, TX, USA; goat anti-human Gal-3 pAb, sc-19280) and actin (Santa Cruz Biotechnology, Dallas, TX, USA; goat anti-human actin pAb, sc-1615).

Techniques:

Journal: PLoS ONE

Article Title: Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

doi: 10.1371/journal.pone.0044400

Figure Lengend Snippet: A: paraffin sections of anterior chamber angle from a normal human eye were immunostained with anti-Gal8 antibody. (i) anti-Gal8 IgG reacted intensely with cells on the trabecular beams (arrows) and with cells in the juxtacanalicular portion of TM (arrowheads). Staining was also observed in the ECM of both portions of TM (JCT and CS) and in the wall of Schlemm’s canal. (ii) No staining was observed when the sections were not exposed to the primary antibody. SC : Schlemm’s canal, JCT : juxtacanalicular TM; CS beams : corneoscleral beams. Bar: 25 µm. B: (i) RT-PCR . Total RNA (1.0 µg) from confluent cultures of normal human TM cells was subjected to RT-PCR. The expected 191 bp fragment was amplified using Gal8- specific-primers. In each case, no components were amplified when reaction mixtures lacked reverse transcriptase (RT). (ii) qRT-PCR . Total RNA was subjected to Taq-Man RT-PCR using Gal8 specific primers. Original amplification plots of Gal8 and GAPDH mRNAs genes are shown (Ct 37.37 and 33.38 for Gal8 and GAPDH, respectively). N = 3 for each experiment; all experiments were performed twice using TM cells from two different donors with reproducible results. (iii and iv) Western Blot Analysis . Protein extracts from confluent cultures of normal human TM cells were incubated with lactogel beads and eluted first with sucrose, and then with lactose. Eluted proteins were electrophoresed, the protein blot of the gel was stained with Ponceau S (iii) and was then processed for immunostaining with goat anti-Gal8 (iv). Both the total cell extract ( T ) and the lactose eluate ( L ) contained a major 36-kDa anti-Gal8 reactive component. This component was not detected in the unbound fraction ( UB ) and in the sucrose eluate ( S ).

Article Snippet: For immunostaining, longitudinal tissue sections (5 μm) were deparaffinized and sequentially treated with a basic pH antigen retrieval reagent (R&D systems, Minneapolis, MN), normal horse serum (R&D systems), goat anti-human Gal8 (2 hr, 37°C, R&D Systems; the goat antibody is specific for Gal8 and it does not recognize other galectins including Gal3 or Gal1), biotinylated anti-goat IgG (1 hr, 25°C, R&D Systems), a freshly prepared solution of avidin-biotin-complex (R&D Systems’ Cell and Tissue Staining Kit, 30 min, 25°C) and a diaminobenzidine/H 2 O 2 reagent (R&D systems).

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Amplification, Reverse Transcription, Quantitative RT-PCR, Western Blot, Incubation, Immunostaining

Journal: PLoS ONE

Article Title: Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

doi: 10.1371/journal.pone.0044400

Figure Lengend Snippet: Normal human TM cells were incubated on microtiter wells coated with BSA, fibronectin, or Gal8 in DPBS, in the presence and absence of a function-blocking anti-β 1 integrin antibody (JB1A), or control mouse IgG. Following incubation at 37°C for 30 min, cells were fixed and stained with crystal violet. Attached cells in fibronectin-coated wells are set as 100% (positive control); attached cells in other wells are presented as percent of positive control. Data are expressed as mean±SEM and analyzed with one-way ANOVA. * P <0.05 vs IgG; ** P <0.01 vs media or IgG; *** P <0.001 vs media. B and C: Cell spreading assay. TM cells were fixed with 4% paraformaldehyde after adhesion for 30 min. F-actin was stained with rhodamine-labeled phalloidin and cell nuclei were labeled with DAPI. Random fields of each experimental condition were photographed, and spread areas of individual cells were quantified with ImageJ. Representative micrographs of TM cells incubated in the presence and the absence of anti-β 1 integrin antibody are shown in C. Data are presented as Box–whisker plot (after Tukey) and analyzed with one-way ANOVA. *** P <0.001 vs media or IgG. This experiment was performed three times with reproducible results. Bar: 100 µm.

Article Snippet: For immunostaining, longitudinal tissue sections (5 μm) were deparaffinized and sequentially treated with a basic pH antigen retrieval reagent (R&D systems, Minneapolis, MN), normal horse serum (R&D systems), goat anti-human Gal8 (2 hr, 37°C, R&D Systems; the goat antibody is specific for Gal8 and it does not recognize other galectins including Gal3 or Gal1), biotinylated anti-goat IgG (1 hr, 25°C, R&D Systems), a freshly prepared solution of avidin-biotin-complex (R&D Systems’ Cell and Tissue Staining Kit, 30 min, 25°C) and a diaminobenzidine/H 2 O 2 reagent (R&D systems).

Techniques: Incubation, Blocking Assay, Control, Staining, Positive Control, Labeling, Whisker Assay

Journal: PLoS ONE

Article Title: Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

doi: 10.1371/journal.pone.0044400

Figure Lengend Snippet: A: Normal human TM cells were plated on eight-chamber glass slides coated with 20 µg/ml of recombinant human Gal8 (i–iii), 20 µg/ml of fibronectin (iv–vi), or 100 µg/ml of poly-L-lysine (vii–ix) in serum-free DMEM at 37°C for 30 min (i, iv, vii), 1 hr (ii, v, viii), and 2 hr (iii, vi, ix). Following the incubation period, cells were fixed with 4% paraformaldehyde and stained with rhodamine-labeled phalloidin. Bar: 50 µm. B: Quantification of stress fiber formation. Random fields were photographed, and cells with robust stress fibers were counted. N = 225 to 362. Data are expressed as mean±SEM and analyzed with one-way ANOVA. *** P <0.001 vs poly-L-lysine at different time points. This experiment was performed three times with reproducible results.

Article Snippet: For immunostaining, longitudinal tissue sections (5 μm) were deparaffinized and sequentially treated with a basic pH antigen retrieval reagent (R&D systems, Minneapolis, MN), normal horse serum (R&D systems), goat anti-human Gal8 (2 hr, 37°C, R&D Systems; the goat antibody is specific for Gal8 and it does not recognize other galectins including Gal3 or Gal1), biotinylated anti-goat IgG (1 hr, 25°C, R&D Systems), a freshly prepared solution of avidin-biotin-complex (R&D Systems’ Cell and Tissue Staining Kit, 30 min, 25°C) and a diaminobenzidine/H 2 O 2 reagent (R&D systems).

Techniques: Recombinant, Incubation, Staining, Labeling

Journal: PLoS ONE

Article Title: Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

doi: 10.1371/journal.pone.0044400

Figure Lengend Snippet: A and C: Serum-starved human TM cells were incubated on chamber glass slides coated with recombinant human Gal8 in the presence of the Rho inhibitor, C3 transferase or ROCK inhibitor, Y27632, at different concentrations. After 2 hr, cells were stained with rhodamine-labeled phalloidin, and cells with robust stress fibers were enumerated. Data are expressed as mean±SEM. B and D: Cells were treated C3 transferase at 2 µg/ml (B) or Y27632 at 20 µM (D), stained with rhodamine-labeled phalloidin, and random fields were photographed. Note that cells treated with Y27632 or C3 transferase are not spread and exhibit dendrite-like structures. Bar: 50 µm.

Article Snippet: For immunostaining, longitudinal tissue sections (5 μm) were deparaffinized and sequentially treated with a basic pH antigen retrieval reagent (R&D systems, Minneapolis, MN), normal horse serum (R&D systems), goat anti-human Gal8 (2 hr, 37°C, R&D Systems; the goat antibody is specific for Gal8 and it does not recognize other galectins including Gal3 or Gal1), biotinylated anti-goat IgG (1 hr, 25°C, R&D Systems), a freshly prepared solution of avidin-biotin-complex (R&D Systems’ Cell and Tissue Staining Kit, 30 min, 25°C) and a diaminobenzidine/H 2 O 2 reagent (R&D systems).

Techniques: Incubation, Recombinant, Staining, Labeling

Journal: PLoS ONE

Article Title: Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

doi: 10.1371/journal.pone.0044400

Figure Lengend Snippet: A: Gal8 induces phosphorylation of MLC2 in a time-dependent manner. Normal human TM cells were incubated on 100-mm dishes coated with Gal8 for 0.5, 1, and 2 hr. Following incubation, cells were lysed, and protein extracts were subjected to electrophoresis in 12% SDS-PAGE gels. Blots were probed with anti-phosphorylated myosin light chain 2 (ppMLC2) (Thr18/Ser19) antibody. The blots were subsequently stripped and reprobed with anti-MLC antibody. A representative Western blot is shown in the top panel. Images were acquired by Odyssey Infrared Imaging System, and band intensity was quantified by ImageJ (bottom panel). N = 3. B: Phosphorylation of MLC2 is inhibited by Rho and ROCK inhibitors. Normal human TM cells were serum-starved overnight and treated with C3 transferase (2 µg/ml) and Y27632 (20 µM) for 4 hr. Treated cells were detached and plated on Gal8-coated dishes for 2 hr in the presence or absence of inhibitors and were then examined for the expression levels of ppMLC2 as described in the legend to panel A. Top: A representative Western blot; bottom: quantification of ppMLC2. Data are expressed as mean±SEM and analyzed with one-way ANOVA. *** P <0.001 vs control. N = 3.

Article Snippet: For immunostaining, longitudinal tissue sections (5 μm) were deparaffinized and sequentially treated with a basic pH antigen retrieval reagent (R&D systems, Minneapolis, MN), normal horse serum (R&D systems), goat anti-human Gal8 (2 hr, 37°C, R&D Systems; the goat antibody is specific for Gal8 and it does not recognize other galectins including Gal3 or Gal1), biotinylated anti-goat IgG (1 hr, 25°C, R&D Systems), a freshly prepared solution of avidin-biotin-complex (R&D Systems’ Cell and Tissue Staining Kit, 30 min, 25°C) and a diaminobenzidine/H 2 O 2 reagent (R&D systems).

Techniques: Phospho-proteomics, Incubation, Electrophoresis, SDS Page, Western Blot, Imaging, Expressing, Control

Journal: BioTechniques

Article Title: Isolation of urinary extracellular vesicles from Tamm- Horsfall protein-depleted urine and their application in the development of a lectin-exosome-binding assay.

doi: 10.2144/000114208

Figure Lengend Snippet: Figure 1. THP in fractions separated during isolation of urinary EVs. (A) Representative SDS-PAGE of pellets (P) and supernatants (S) separated by salt precipitation of urine at 3000 × g (P3000, S3000), 17,000 × g (P17000, S17000), and 100,000 × g (P100000, S100000). The broad strong band at 110 kDa corresponding to THP monomer was detected for the 3000 × g pellet (P3000), whereas a weak band at the same position was detected for the 17,000 × g pellet (P17000). (B) Matching fractions separated from the same urine sample without salt precipitation had different protein compositions, and the THP band was evident in the uEV preparation (Figure 1B). The gels were silver-stained. Numbers indicate the position of molecular mass standards (MWSt) in kDa. (C) Representative immunoblot of the 100,000 × g P containing pure uEVs. uEV proteins were transferred onto a membrane and probed with monoclonal anti-CD63 antibody and polyclonal anti-gal-3 antibodies. (D) Transmission electron micrographs of uEVs isolated from THP-depleted urine by salt precipitation. Isolated uEVs were of typical size and shape and did not differ between male (a) and female (b) donors.

Article Snippet: Goat anti-human galectin-3 (gal-3) antibody was obtained from R&D Systems (Minneapolis, MN).

Techniques: Isolation, SDS Page, Staining, Western Blot, Membrane, Transmission Assay

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