Journal: Journal of the American Heart Association

Article Title: Smooth Muscle Cell‐Derived Fibronectin Promotes an Atheroprotective Smooth Muscle Cell Phenotype Associated With Altered NO‐cGMP Signaling

doi: 10.1161/jaha.124.040395

Figure Lengend Snippet: Figure 3. Analysis of modulated SMCs in atherosclerotic aortae. A, UMAP visualization of selected cell clusters (SMC 2, mod-SMC, and MΦ) from atherosclerotic aortae of control and Fn-smko mice shown in Figure 2A. B, Distribution of cells expressing Gucy1a1 (left bar graph) and Gucy1b1 (right bar graph) mRNA across the 3 clusters shown in A. C, Representative Western blot detection (left) and semiquantitative analysis (right bar graphs) of Fn1, Gucy1b1, Prkg1, and Acta2 in atherosclerotic aortae from 1-year-old control (n=3) and Fn-smko (n=3) mice on chow diet. Gapdh was used as a loading control. The position of molecular weight markers in kDa is shown next to the Western blots. D, Immunostaining of Gucy1b1 of aortic arch sections from mice fed 18 weeks with HFD (left, black arrows indicate examples of positive cells) and quantification of Gucy1b1+ cells in the lesion core (right bar graph) (control n=12 lesions from 4 mice; Fn-smko n=16 lesions from 6 mice). E, Immunostaining of Lgals3 in parallel sections of those shown in (D) (left, black arrows indicate some positive cells) and quantification of Lgals3+ cells in the lesion core (right bar graph) (control n=23 lesions from 6 mice; Fn-smko n=27 lesions from 7 mice). Scale bars in (D) and (E), 100 μm. Black lines in (D) and (E) indicate the lesion with the fibrous cap outlined as the 30-μm layer of the lesion adjacent to the vascular lumen. F, Plaque stability determined by the ratio of Acta2+ over Lgals3+ lesion area measured in parallel sections (control n=25 lesions from 6 mice; Fn-smko n=27 lesions from 7 mice). Note that the control group contained 1 data point >5. This data point is not shown in the bar graph but included in statistical analysis. Data are shown as mean±SEM. *P<0.05 by unpaired t test (C) or Mann–Whitney U test (D through F). Acta2 indicates alpha smooth muscle actin; C, core; Ctrl, control; FC, fibrous cap; Fn-smko, smooth muscle-specific deletion of fibronectin; Gucy1a1, alpha1 subunit of NO-GC; Gucy1b1, beta1 subunit of NO-GC; HFD, high-fat diet; Lgals3, galectin 3; MΦ, macrophage; mod-SMC, modulated SMC; Prkg1, cGMP-dependent protein kinase I; SMC, smooth muscle cell; and UMAP, uniform manifold approximation and projection.

Article Snippet: Paraffin- embedded sections of left anterior descending arteries from previously described autopsy material38 were stained with hematoxylin/ eosin or immunostained with antibodies recognizing human LGALS3 (R&D Systems, AF1154, from goat) or GUCY1B1 (Abcam, ab24824, from rabbit).

Techniques: Control, Expressing, Western Blot, Molecular Weight, Immunostaining, MANN-WHITNEY

Journal: Journal of the American Heart Association

Article Title: Smooth Muscle Cell‐Derived Fibronectin Promotes an Atheroprotective Smooth Muscle Cell Phenotype Associated With Altered NO‐cGMP Signaling

doi: 10.1161/jaha.124.040395

Figure Lengend Snippet: Figure 4. NO-GC expression in modulated SMCs of murine atherosclerotic plaques. A, Immunofluorescence staining of Gucy1b1 (red) and Lgals3 (green) in an atherosclerotic lesion. Scale bars, 100 μm. The framed area is shown at higher magnification in the adjacent image on the right. White arrowheads indicate Gucy1b1/Lgals3 coexpressing cells (yellow). Scale bar, 10 μm. B, Lineage tracing experiment (for experimental details, see Methods section). SMCs in the aortic media were genetically pulse labeled before the onset of atherosclerosis resulting in permanent expression of β-galactosidase in the originally labeled cells as well as in their progeny. After the development of atherosclerotic plaques, SMC-derived cells were visualized by X-Gal staining of the aorta (blue). Lgals3 (brown, left) and Gucy1b1 (brown, right) were detected by immunohistochemical staining of X-Gal-stained plaque sections. Scale bars, 100 μm. The framed areas are shown at higher magnification in the adjacent images on the right. Examples of SMC-derived cells expressing Lgals3 or Gucy1b1 (blue and brown) are indicated by arrowheads. Scale bars, 25 μm. Atherosclerotic aortae analyzed in (A) and (B) were from- 52-week-old apolipoprotein E-deficient mice fed with a normal chow. Shown immunostainings are representative for 3 independent experiments. C, UMAP visualization of modulated SMCs isolated from atherosclerotic aortae shown in Figure 2A. Upper sections show cells with detectable Gucy1a1 (red) and Gucy1b1 (green) transcripts. Middle sections show cells expressing Gucy1a1 (red) and Lgals3 (green) mRNA, and lower sections show cells expressing Gucy1b1 (red) and Lgals3 (green) mRNA. Overlays (right) show coexpressing cells in yellow. C indicates core; FC, fibrous cap (30-μm layer of the lesion adjacent to the vascular lumen); Gucy1a1, alpha1 subunit of NO-GC; Gucy1b1, beta1 subunit of NO-GC; Lgals3, galectin 3; SMC, smooth muscle cell; and UMAP, uniform manifold approximation and projection.

Article Snippet: Paraffin- embedded sections of left anterior descending arteries from previously described autopsy material38 were stained with hematoxylin/ eosin or immunostained with antibodies recognizing human LGALS3 (R&D Systems, AF1154, from goat) or GUCY1B1 (Abcam, ab24824, from rabbit).

Techniques: Expressing, Immunofluorescence, Staining, Labeling, Derivative Assay, Immunohistochemical staining, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Proteomic Profiling of Two Distinct Populations of Extracellular Vesicles Isolated from Human Seminal Plasma

doi: 10.3390/ijms21217957

Figure Lengend Snippet: Isolation and characterization of LEV and SEV. Total EV in seminal plasma of vasectomized men were collected by UC at the interface of a sucrose block gradient. LEV and SEV were separated by their distinct velocities during upward displacement into a continuous sucrose density gradient. ( A ) Gradient fractions were analyzed by SDS-PAGE followed by Sypro ruby staining for total protein. ( B ) Gradient fractions were analyzed by immunoblotting for the presence of EV associated proteins, including CD9, CD81, PSCA, Galectin-3, CD63, HSP70, Annexin A1, and GLIPR2. Molecular weight markers are indicated on the left in kDa. ( C ) Particles in the LEV and SEV containing fractions were analyzed by TEM. Scale bar, 500 nm. Arrows exemplify incidental SEV in the LEV isolate. ( D ) Size distribution of the particles in LEV and SEV isolates as determined by NTA.

Article Snippet: The primary antibodies used were mouse anti human CD9 (HI9a, 312102, Biolegend, San Diego, California, USA, 1:2000); mouse anti human CD81 (B11, sc-166029, Santa Cruz Biotechnology, Dallas, Texas, USA, 1:500); mouse anti human HSP70 (N27F3-4, ADI-SPA-820-D, Enzo, Bruxelles, Belgium, 1:1000); mouse anti human Flotillin-1 (clone 18, 610821, BD Biosciences, San Jose, California, USA, 1:1000); mouse anti-human PSCA (clone 7F5, sc-80654, Santa Cruz Biotechnology, Dallas, Texas, USA, 1:1000); mouse anti human Annexin A1 (clone 29, 610066, BD Biosciences, San Jose, California, USA, 1:1000); mouse anti human CD47 (B6H12, sc-12730, Santa Cruz Biotechnology, Dallas, Texas, USA, 1:500); rat anti human galectin-3 (M3/38, CL8942B, CEDARLANE, Burlington, Vermont, USA, 1:500) and rabbit anti human GLIPR2/GAPR-1 (Eberle et al., 2002, 1:5000, kindly provided by J.B. Helms).

Techniques: Isolation, Blocking Assay, SDS Page, Staining, Western Blot, Molecular Weight

Journal: Molecular Oncology

Article Title: Extracellular LGALS3BP : a potential disease marker and actionable target for antibody–drug conjugate therapy in glioblastoma

doi: 10.1002/1878-0261.13453

Figure Lengend Snippet: LGALS3BP expression levels in GBM patients. (A) Sandwich ELISA performed on GBM patient ( n = 17) and healthy donor ( n = 18) serum samples. (B) Graph showing the percentage of EVs‐associated LGALS3BP over total circulating LGALS3BP in serum. Mann–Whitney test * P = 0.03. (C) Box‐and‐whisker diagram of the distribution of LGALS3BP in GBM cases and peritumoral matched samples ( n = 53). The upper and lower ends of boxes represent 75th and 25th percentiles. The median value is shown with a solid line. Mann–Whitney test *** P < 0.001. Data are shown as mean ± standard deviation. (D, E) Representative images of IHC staining for LGALS3BP expression in GBM and peritumoral matched samples. All IHC images have been acquired using an optical microscope at a scale bar of 50 μm.

Article Snippet: Also, for LGALS3BP expression analysis of mice serum‐derived EVs in correlation studies, they were analysed using Proteintech™ Human LGALS3BP ELISA Kit (Cod.

Techniques: Expressing, Sandwich ELISA, MANN-WHITNEY, Whisker Assay, Standard Deviation, Immunohistochemistry, Microscopy

Journal: Molecular Oncology

Article Title: Extracellular LGALS3BP : a potential disease marker and actionable target for antibody–drug conjugate therapy in glioblastoma

doi: 10.1002/1878-0261.13453

Figure Lengend Snippet: LGALS3BP expression levels in EVs isolated from GBM patient‐derived cell lines. (A) LGALS3BP and EVs markers CD9, actin and CD63 expression levels in whole lysate and EVs isolated from Gch6 and Gch14 cellular supernatants ( n = 2). (B) Effect of N‐Glycosylation (kifunensine, KIF and tunicamycin, TUN) and O‐Glycosylation (OSMI‐1) inhibitors on the protein profile for LGALS3BP in Gch6 whole lysate and corresponding supernatant ( n = 2). (C) Sandwich ELISA performed on intact EVs isolated from Gch6 and Gch14 cell line supernatants ( n = 3). Data are shown as mean ± standard deviation. (D) Sandwich ELISA performed on intact EVs isolated from Gch6 treated with KIF ( n = 3). Data are shown as mean ± standard deviation. (E) Effect of KIF treatment on the protein profile in whole lysate and EVs isolated from Gch6 ( n = 2). (F) Confocal images of live GBM patient‐derived cells labelled with humanized 1959 anti‐LGALS3BP antibody followed by AlexaFluor 488 conjugated secondary anti‐human IgG antibody (green). Cell nuclei were stained with DAPI (blue) ( n = 3). Images were taken at 40× magnification. Scale bar: 20 μm. Negative controls were only incubated with secondary antibody.

Article Snippet: Also, for LGALS3BP expression analysis of mice serum‐derived EVs in correlation studies, they were analysed using Proteintech™ Human LGALS3BP ELISA Kit (Cod.

Techniques: Expressing, Isolation, Derivative Assay, Sandwich ELISA, Standard Deviation, Staining, Incubation

Journal: Molecular Oncology

Article Title: Extracellular LGALS3BP : a potential disease marker and actionable target for antibody–drug conjugate therapy in glioblastoma

doi: 10.1002/1878-0261.13453

Figure Lengend Snippet: Extracellular vesicles‐associated LGALS3BP correlation with tumour volume and therapeutic activity of 1959‐sss/DM3 in GBM patient‐derived xenograft model. (A) Graphic scheme representing ELISA of human LGALS3BP on EVs in serum samples from mice with human GBM xenograft. (B) Levels of LGALS3BP on EVs isolated from the serum samples of control nude mice or mice bearing human GCh6 GBM xenograft, measured by ELISA ( n = 5). Bar graph represents average ± standard deviation. (C) Spearman correlation between EVs‐associated LGALS3BP and tumour burden in xenograft‐bearing nude mice ( n = 9). (D, E) Cytotoxic activity of SH‐DM3, SH‐DM4 and temozolomide (TMZ) was obtained by using the MTT assay on Gch6 and Gch14 cell lines at indicated doses after 5 days. IC50 values were determined with graphpad prism software (San Diego, CA, USA) ( n = 3). Data are shown as mean ± standard deviation. (F) CD1 nude mice harbouring Gch6 xenografts were treated with vehicle (PBS, n = 4) or 1959‐sss/DM3 (10 mg·kg −1 , n = 4) twice weekly as indicated by the arrows. Data are shown as mean ± standard error. (G) (PBS, n = 4) (10 mg·kg −1 , n = 4). Kaplan–Meier survival curves. Log‐rank (Mantel–Cox) Test. ** P = 0.006.

Article Snippet: Also, for LGALS3BP expression analysis of mice serum‐derived EVs in correlation studies, they were analysed using Proteintech™ Human LGALS3BP ELISA Kit (Cod.

Techniques: Activity Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Isolation, Standard Deviation, MTT Assay, Software

Journal: Molecular Oncology

Article Title: Extracellular LGALS3BP : a potential disease marker and actionable target for antibody–drug conjugate therapy in glioblastoma

doi: 10.1002/1878-0261.13453

Figure Lengend Snippet: 1959‐sss/DM4 dose–response efficacy and biodistribution. (A) CD1 nude mice Gch6 xenografts were treated with vehicle (PBS) or 1959‐sss/DM4 at the indicated doses twice weekly (PBS, n = 5; 1 mg·kg −1 , n = 7; 3 mg·kg −1 , n = 6; 10 mg·kg −1 , n = 6). Data are shown as mean ± standard error. (B) (PBS, n = 5; 1 mg·kg −1 , n = 7; 3 mg·kg −1 , n = 6; 10 mg·kg −1 , n = 6). Kaplan–Meier survival curves. Log‐rank (Mantel–Cox) Test. *** P < 0.001. (C) Representative images of IHC staining for LGALS3BP expression in Gch6 patient primary tumour and in Gch6 xenograft ( n = 4). Scale bar: 50 μm. (D) Biodistribution of 1959‐sss/DM4 in tumour‐bearing nude mice. ADC was injected into the tail vein of nude mice bearing a xenograft of GCh6 patient‐derived cell line. After 24, 48 and 72 h, the mice were sacrificed, and the ADC uptake was determined in tissues (D) by ELISA using an anti‐DM4 antibody as coating. Each column and bar show the mean and standard deviation for three mice. Tissue uptake of ADC is expressed as percentage of injected dose per gram of tissue (%ID·g −1 ). Data are shown as mean ± standard deviation.

Article Snippet: Also, for LGALS3BP expression analysis of mice serum‐derived EVs in correlation studies, they were analysed using Proteintech™ Human LGALS3BP ELISA Kit (Cod.

Techniques: Immunohistochemistry, Expressing, Injection, Derivative Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Development of galectin-3 knockdown ARPE-19 cells. ( A , B ) Merged phase-contrast and fluorescent imaging for GFP expression of ARPE-19 cells without ( A ) and with pCRISPR-LGALS3 transfection ( B ). ( C , D ) Immunostaining for galectin-3 of ARPE-19 ( C ) and ARPE-19/LGLAS3 +/− cells ( D ) after FACS and single-cell cultivation. ( E , F ) Phase-contrast image of ARPE-19 ( E ) and ARPE-19/LGLAS3 +/− cells ( F ). Magnification bar in ( A , B ) 100 µm; in ( C , D ) 20 µm, in ( E , F ) 100 µm; blue, Hoechst staining. ( G ) Western blot analysis for galectin-3 of ARPE-19 and ARPE-19/LGLAS3 +/− cells. ( H , I ) Galectin-3 concentration of conditioned cell culture medium ( H ) and cell lysate ( I ) from ARPE-19 and ARPE-19/LGLAS3 +/− cells after incubation in unsupplemented cell culture medium for 72 h. Mean ± SD; ** p < 0.01; **** p < 0.0001; n = 10.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Knockdown, Imaging, Expressing, Transfection, Immunostaining, Staining, Western Blot, Concentration Assay, Cell Culture, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Reduced expression of galectin-3 decreases viability of immortalized RPE cells in vitro. WST-1 assay of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells following incubation in cell culture medium without supplementation and various concentrations of hr-galectin-3 (hr-Gal3) for 72 h. Mean ± SD; **** p < 0.0001; n ≥ 28 of at least 4 independent experiments.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, In Vitro, WST-1 Assay, Incubation, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Decreased expression of galectin-3 declines proliferation of immortalized RPE cells in vitro. BrdU ELISA of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells following incubation in cell culture medium without supplementation for 72 h with and without various concentrations of hr-galectin-3 ( A ) and/or an additional treatment with 100mM lactose ( B ). Mean ± SD; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; n ≥ 20 of at least 4 independent experiments.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Incubation, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Decreased galectin-3 expression reduces migration of ARPE-19 cells in vitro. Quantification ( A ) and representative images ( B ) of scratch migration assay of native ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells following incubation with and without hr-galectin-3 in cell culture medium without supplements for 24 h. ( A ) For quantification, the recolonized area after 24 h was calculated and plotted as the relative recolonized area. ( B ) Representative images of native ARPE-19 (left panel) and ARPE-19/LGALS3 +/− cells (right panel) immediately after scratching (0 h, upper row) and after incubation for 24 h (lower panel). Mean ± SD; *** p < 0.001; **** p < 0.0001; n ≥ 10 of 3 independent experiments.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, Migration, In Vitro, Incubation, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Reduced expression of endogenous galectin-3 enhances cell attachment of immortalized RPE cells in vitro. ( A ) Cell adhesion of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells was quantified 30, 60, 90, 120, 150, and 180 min after seeding and plotted as the relative number of adherent cells. Mean ± SD; n = 12 for each group of 3 independent experiments; * comparison of ARPE-19 versus ARPE-19/LGALS3 +/− cells; # comparison of ARPE-19/FACS versus ARPE-19/LGALS3 +/− cells; **** p < 0.001; #### p < 0.001. ( B ) Relative number of adherent ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells 90 min after seeding and incubation with various concentrations of hr-galectin-3, which was added immediately before seeding. Mean ± SD; n = 15 for each group of 3 independent experiments; ** p < 0.01; **** p < 0.0001.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, Cell Attachment Assay, In Vitro, Comparison, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Lack of endogenous galectin-3 promotes epithelial-to-mesenchymal transition of immortalized RPE. ( A – F ) Phaloidin staining (( A , B ) orange) as well as immunofluorescent staining for sm-α-actin (( C , D ) orange) and N-cadherin ( E , F ) green) of ARPE-19 ( A , C , E ) and ARPE-19/LGALS3 +/− cells ( B , D , F ). Scale bar, 20 µm; blue, DAPI staining. ( G ) Real-time rt-PCR for sm-α-actin, E-cadherin, and N-cadherin mRNA expression of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells. Mean ± SD; n ≥ 7 of 5 independent experiments; * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Staining, Quantitative RT-PCR, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Role of Endogenous Galectin-3 on Cell Biology of Immortalized Retinal Pigment Epithelial Cells In Vitro †

doi: 10.3390/ijms26157622

Figure Lengend Snippet: Endogenous galectin-3 expression maintains basal pAKT, pERK, and β-catenin signaling in immortalized RPE cells. Western blot analysis ( A ) and densitometry for pAKT ( B ), pERK ( C ), and β-catenin ( D ) expression of ARPE-19, ARPE-19/FACS, and ARPE-19/LGALS3 +/− cells. Mean ± SD; n ≥ 6 of 6 independent experiments; * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Galectin-3 knockdown ARPE-19 cells were generated using the all-in-one sgRNA pCRISPR plasmid against human LGALS3 (pCRISPR-LGALS3; HCP301784-CG04-3-Bc, Genecopoeia, Rockville, MA, USA).

Techniques: Expressing, Western Blot