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: Investigative Ophthalmology & Visual Science

Article Title: Diurnal Photoreceptor Outer Segment Renewal in Mice Is Independent of Galectin-3

doi: 10.1167/iovs.62.2.7

Figure Lengend Snippet: Gal-3 localizes to the neural retina and RPE in mice. ( A ) Immunofluorescence staining of gal-3 of retinal sections from wt and lgals3 −/− mice shows localization to the inner retina (and they are based on the appearance most likely to be mainly Müller cells), RPE, and choroid. Images are representative maximal projections. Green : gal-3; and blue : nuclei counterstain. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, photoreceptor outer segment layer. Scale bar = 50 µm. ( B ) Magnified fields of stained sections as in A focusing on the RPE. Scale bar = 10 µm. ( C ) Immunofluorescence staining of RPE whole mount preparations of wt and lgals3 −/− mice show gal-3 in the RPE. Images are representative maximal projections comprising the entire RPE. Green : gal-3; red : F-actin; blue : nuclei counterstain. Scale bar = 25 µm.

Article Snippet: Primary antibodies were to: rhodopsin B630, blue opsin (Millipore-Sigma; #5407), red/green opsin (Millipore-Sigma; #5405), GAPDH (Genetex, Irvine, CA, USA; #GTX100118), gal-3, MerTK (R&D Systems; #AF591), RPE65 (Genetex; #GTX103472), and anti-PSD95 (Cell Signaling, Danvers, MA, USA; #S4507).

Techniques: Immunofluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Diurnal Photoreceptor Outer Segment Renewal in Mice Is Independent of Galectin-3

doi: 10.1167/iovs.62.2.7

Figure Lengend Snippet: Unlike degenerating mertk − / − retina, lgals3 − / − retina has wt levels of RPE and photoreceptor marker proteins, but gal-3 is elevated in posterior eyecups of mertk − / − mice with retinal degeneration. ( A ) Representative comparative immunoblots of detergent lysates representing equal fractions of whole eyes without lens with intact retinas of 3-month-old wt, lgals3 −/− , and mertk −/− mice probed for photoreceptor and RPE marker proteins and housekeeping proteins GAPDH as loading control as indicated. Probes for gal-3 and MerTK confirmed mouse genotypes. ( B ) Bars show quantification of average rhodopsin and cone opsin content in lgals3 − / − ( black bars ) and mertk − / − eyes ( white bars ) normalized to content in wt eyes as indicated. ( C ) Representative immunoblots of posterior eyecups (ECs) and neural retina (NR) eye fractions probed for proteins as indicated. Probes for RPE65, PSD95, and opsins confirmed fraction enrichment with minor contamination of adjacent tissue as expected of manual dissections. GAPDH indicated protein load. ( D ) Bars show quantification of gal-3 in mertk −/− eye fractions ( white bars ) and of MerTK in lgals3 −/− eye fractions ( black bars ), respectively. Bars in B and D show averages ± s. e. m., n = 4; Student's t -test; * p < 0.05. There was no difference in levels of opsins, MerTK or any other marker tested between lgals3 −/− and wt tissue. As expected, mertk −/− eyes with ongoing retinal degeneration contained dramatically reduced levels of opsins and other neural retina markers but normal levels of RPE marker RPE65 and elevated levels of gal-3.

Article Snippet: Primary antibodies were to: rhodopsin B630, blue opsin (Millipore-Sigma; #5407), red/green opsin (Millipore-Sigma; #5405), GAPDH (Genetex, Irvine, CA, USA; #GTX100118), gal-3, MerTK (R&D Systems; #AF591), RPE65 (Genetex; #GTX103472), and anti-PSD95 (Cell Signaling, Danvers, MA, USA; #S4507).

Techniques: Marker, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Diurnal Photoreceptor Outer Segment Renewal in Mice Is Independent of Galectin-3

doi: 10.1167/iovs.62.2.7

Figure Lengend Snippet: Purified gal-3 does not promote binding or engulfment of outer segment particles by primary mouse RPE cells. Unpassaged polarized mouse RPE cells were fed with Texas Red-labeled POS in medium with no additional protein ( A , B , top rows ), added protein S ( A , B , center rows ), or added gal-3 ( A , B , bottom rows ). All samples received integrin ligand MFG-E8 to promote POS binding. Bound or engulfed POS material is shown in red. Bound particles only were counterstained post-fixation with rhodopsin antibody ( green ). Nuclei were counterstained in blue . Apically bound particles ( green and red ) are shown in apical maximal projections in A . Engulfed particles of the same fields as in A are shown in B in maximal projections of the center of the RPE as indicated by the RPE nuclei. Scale bar = 25 µm. ( C , D ) Bar graphs shows quantification of bound POS C and internalized POS D , respectively, relative to samples with only MFG-E8 added, which was set as 1. The bona fide MerTK ligand Protein S was added at 2 µg/mL ( gray bars ) to serve as positive control for MerTK-dependent POS internalization. Gal-3 was added at 2.5, 5, or 10 µg/mL ( black bars , concentrations as indicated). Bars show mean ± s. e. m., n = 4 independent assays; 1-way ANOVA with Tukey's post hoc test; * p < 0.05. Differences between samples fed with MFG-E8 only or with MFG-E8 plus gal-3 at any concentration tested were not significant for either bound or internalized POS.

Article Snippet: Primary antibodies were to: rhodopsin B630, blue opsin (Millipore-Sigma; #5407), red/green opsin (Millipore-Sigma; #5405), GAPDH (Genetex, Irvine, CA, USA; #GTX100118), gal-3, MerTK (R&D Systems; #AF591), RPE65 (Genetex; #GTX103472), and anti-PSD95 (Cell Signaling, Danvers, MA, USA; #S4507).

Techniques: Purification, Binding Assay, Labeling, Positive Control, Concentration Assay

Journal: Cardiovascular Diagnosis and Therapy

Article Title: Galectin-3 promotes calcification of human aortic valve interstitial cells via the NF-kappa B signaling pathway

doi: 10.21037/cdt-21-506

Figure Lengend Snippet: Upregulation of Gal-3 in blood and AVs of CAVD patients. (A) Serum Gal-3 concentration in CAVD and control subjects (n=20); (B) mRNA expression levels of Gal-3 in AVs from CAVD patients and control subjects (n=4); (C) quantification of western blot; (D) protein expression levels of Gal-3 in AVs from CAVD patients and control subjects (n=5); (E) representative pictures of AV sections immunostained for Gal-3 and ALP observed at low (×5, scale bar: 1 mm) and high (×20, scale bar: 100 µm) magnification in controls and CAVD patients. Statistical comparisons were made using Student’s t-test. All of the data are presented as mean ± SEM. *, P<0.05, **, P<0.01. Gal-3, galectin-3; AVs, aortic valves; CAVD, calcific aortic valve disease.

Article Snippet: After blocking with 5% non-fat dry milk in 0.05% Tween 20 for 1 h at room temperature, the blotted membranes were incubated with primary antibodies overnight for Runx2 (CST, 8486 s), ALP (R&D, AF2910), Gal-3 (Zenbio, 250013), GAPDH (Proteintech, 60004-1-Ig), IκBα (CST, 4814 s), and p-IκBα (CST, 2859 s) at 4 ℃.

Techniques: Concentration Assay, Expressing, Western Blot

Journal: Cardiovascular Diagnosis and Therapy

Article Title: Galectin-3 promotes calcification of human aortic valve interstitial cells via the NF-kappa B signaling pathway

doi: 10.21037/cdt-21-506

Figure Lengend Snippet: Effect of Gal-3 on calcification-related gene/protein expression and osteogenic differentiation of hVICs. (A) hVICs interfered with or without 2 µg/mL Gal-3 for 72 h, and the mRNA expression levels of ALP and Runx2 detected by qRT-PCR (n=4); (B) Western blotting of Gal-3-treated ALP and Runx2 proteins; (C) quantification of western blot (n=4); (D) Alizarin Red S staining of the cells under different conditioned treatments, including control (normal culture medium), OM (osteogenic medium), and OM + Gal-3 (osteogenic medium with Gal-3 treatment). The microscopic images were taken with the 4× objective; (E) quantification of alizarin red positive area (n=3); (F) immunofluorescent staining was carried out to detect ALP in hVICs treated with or without Gal-3 (scale bar: 50 µm). Statistical comparisons were made using Student’s t-test. All of the data are presented as mean ± SEM. *, P<0.05, **, P<0.01. Gal-3, galectin-3; hVICs, human aortic valve interstitial cells; qRT-PCR, quantitative real-time polymerase chain reaction.

Article Snippet: After blocking with 5% non-fat dry milk in 0.05% Tween 20 for 1 h at room temperature, the blotted membranes were incubated with primary antibodies overnight for Runx2 (CST, 8486 s), ALP (R&D, AF2910), Gal-3 (Zenbio, 250013), GAPDH (Proteintech, 60004-1-Ig), IκBα (CST, 4814 s), and p-IκBα (CST, 2859 s) at 4 ℃.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Real-time Polymerase Chain Reaction

Journal: Cardiovascular Diagnosis and Therapy

Article Title: Galectin-3 promotes calcification of human aortic valve interstitial cells via the NF-kappa B signaling pathway

doi: 10.21037/cdt-21-506

Figure Lengend Snippet: Gene expression profile by RNA-sequencing of hVICs cultured with or without Gal-3. (A) Volcano map of DEGs in C versus Gal-3 [log10 (Gal-3/C); up-regulation: 79 genes and down-regulation: 82 genes]; (B) heatmap for common DEGs with group clusters; (C) KEGG pathway enrichment, where orange dots indicate the degree of enrichment [Q value (−log10)]; (D) transcription factor enrichment, yellow (deep) color indicates the degree of enrichment [Q value (−log10)]. Gal-3, galectin-3; hVICs, human aortic valve interstitial cells; DEGs, differentially expressed genes.

Article Snippet: After blocking with 5% non-fat dry milk in 0.05% Tween 20 for 1 h at room temperature, the blotted membranes were incubated with primary antibodies overnight for Runx2 (CST, 8486 s), ALP (R&D, AF2910), Gal-3 (Zenbio, 250013), GAPDH (Proteintech, 60004-1-Ig), IκBα (CST, 4814 s), and p-IκBα (CST, 2859 s) at 4 ℃.

Techniques: Expressing, RNA Sequencing Assay, Cell Culture

Journal: Cardiovascular Diagnosis and Therapy

Article Title: Galectin-3 promotes calcification of human aortic valve interstitial cells via the NF-kappa B signaling pathway

doi: 10.21037/cdt-21-506

Figure Lengend Snippet: Gal-3 treatment activated NF-κB signaling pathway in hVICs. (A) Western blot analysis revealed the phosphorylation level of IκBα in hVICs treated with Gal-3 for 0, 30, 60, and 120 min; (B) the phosphorylation level of IκBα in hVICs treated with or without Gal-3 or BAY11-7082 for 30 min, assessed by Western blot; (C) quantification of western blot; (D) immunofluorescence of p65 in hVICs treated with or without Gal-3 or BAY11-7082 for 30 min; (E) the protein expression levels of osteogenesis-specific markers (ALP, Runx2) were determined by Western blot in hVICs treated with OM, OM + Gal-3, or OM, Gal-3 + BAY11-7082 for 48 h; (F) quantification of western blot (n=3); (G) the immunofluorescence staining of ALP and Runx2 in AVICs with Gal-3 or Gal-3 + BAY11-7082; (H) representative images of Alizarin Red staining showing calcium deposits in hVICs treated with OM, OM + Gal-3 or OM, Gal-3 + BAY11-7082 for 21 days. The microscopic images were taken with the 4× objective; (I) quantification of alizarin red positive area (n=3). Statistical comparisons were made using Student’s t-test. All of the data are presented as mean ± SEM. *, P<0.05, **, P<0.01, ***, P<0.001. Gal-3, galectin-3; hVICs, human aortic valve interstitial cells.

Article Snippet: After blocking with 5% non-fat dry milk in 0.05% Tween 20 for 1 h at room temperature, the blotted membranes were incubated with primary antibodies overnight for Runx2 (CST, 8486 s), ALP (R&D, AF2910), Gal-3 (Zenbio, 250013), GAPDH (Proteintech, 60004-1-Ig), IκBα (CST, 4814 s), and p-IκBα (CST, 2859 s) at 4 ℃.

Techniques: Western Blot, Immunofluorescence, Expressing, Staining