Journal: Scientific reports

Article Title: Targeting galectin-3 with a high-affinity antibody for inhibition of high-grade serous ovarian cancer and other MUC16/CA-125-expressing malignancies.

doi: 10.1038/s41598-021-82686-3

Figure Lengend Snippet: Figure 1. Expression of LGALS3 and CA125 (MUC16) in human ovarian cancers. Three anonymized human tissue microarrays were constructed by collecting 1–3 cores from paraffin embedded, fixed blocks, as previously described27. (A) Examples of human tumor cores stained for CA125 (panel i) and Gal3 (panel ii). Examples of core that did not stain for CA125 (panel iv) and tumor that did not stain for Gal3 (panel iii). (B) Each core was qualitatively scored for prevalence of CA125 and Gal3 positive cells (0 = < 5%, 1 + = 5–25%; 2 + = 25–50%; 3 + = 50–75% and 4 + = > 75%. The intensity for Gal3 was separately scored as low, medium and high (represented by green, orange and red dots respectively). All cores were independently scored by a blinded reference pathologist (KP).

Article Snippet: The last 2 immunizations used human LGALS3 protein (OriGene; Rockville, MD).

Techniques: Expressing, Construct, Staining

Journal: Frontiers in Immunology

Article Title: Granulosa cell-derived miR-379-5p regulates macrophage polarization in polycystic ovarian syndrome

doi: 10.3389/fimmu.2023.1104550

Figure Lengend Snippet: Increased M1/M2 ratio and galectin-3 suppresses granulosa cell proliferation in antral, but not preantral follicle stage, as evident in PCOS. (A) PCOS subjects had lower M2 population and higher M1/M2 ratio compared to non-PCOS; (B) PCOS subjects had higher FF content of galectin-1 and -3 compared to non-PCOS; (C) Galectin-1 did not affect rat GC proliferation in preantral and antral follicles, galectin-3 significantly reduced GC proliferation in antral but not preantral follicles; (D) Adding PCOS FF to non-PCOS GCs significantly reduced cell proliferation compared to those treated with non-PCOS FF. However, the effect of PCOS FF on GC proliferation was blocked using galectin-3 neutralizing antibody; (E) Adding human recombinant galectin-3 to non-PCOS GCs significantly suppress proliferation; (F) The combination of galectin-3 and Anti-Müllerian hormone (AMH) improve sensitivity and specificity of PCOS prediction compared to AMH. AMH: AUC=0.96 with sensitivity=0.88 and specificity=0.95 (p<0.0001); Galectin-3: AUC=0.84 with sensitivity=0.65 and specificity=0.95 (p<0.001); Galectin-3+AMH: AUC=0.977 with sensitivity=0.94 and specificity=1 (p<0.0001). Results are expressed as mean±SEM (n=3 replicates each with 2 rats/group). Data were analyzed by two-way ANOVA and tukey post hoc. ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Human recombinant galectin-3 (R & D systems; n = 3; 0.03 ng/mL) was added to non-PCOS granulosa cell cultures.

Techniques: Recombinant

Journal: Frontiers in Immunology

Article Title: Granulosa cell-derived miR-379-5p regulates macrophage polarization in polycystic ovarian syndrome

doi: 10.3389/fimmu.2023.1104550

Figure Lengend Snippet: MiR-379-5p-induced M1 polarization regulates granulosa cell proliferation in a follicular stage-specific manner by increasing pro-inflammatory cytokine level. (A) Conditioned media from macrophages transfected by miR379 mimic (CM-mimic) increases aromatase content without affecting proliferation in preantral follicle GC, while it reduces aromatase and proliferation in that of antral follicles. The aromatase and proliferative responses were not significantly influenced by the presence of the CM-inhibitor compared to control in both follicle stages; (B) Profile of cytokines (Proteome Profiler Rat XL Cytokine Array; R&D Systems) in macrophage condition medium indicates that CM-mimic had higher content of inflammatory cytokines, including Cystatin C (5.4 fold), Galectin-3 (5.3 fold), TNFRS11B (4.3 fold), PAI-1 (3.9 fold) and Osteopontin (3.7 fold). In addition, Endostatin, Fibulin 3, TNF-α, Galectin-1, IL-6, Jagged 1 and MMP3 were specific cytokines to CM-mimic. Results are expressed as mean±SEM (n=3 replicates each with 2 rats/group). Data were analyzed by two-way ANOVA and tukey post hoc. * P <0.05, *** P <0.001.

Article Snippet: Human recombinant galectin-3 (R & D systems; n = 3; 0.03 ng/mL) was added to non-PCOS granulosa cell cultures.

Techniques: Transfection

Journal: Frontiers in Immunology

Article Title: Granulosa cell-derived miR-379-5p regulates macrophage polarization in polycystic ovarian syndrome

doi: 10.3389/fimmu.2023.1104550

Figure Lengend Snippet: Hypothetical model illustrating that uptake of GC-derived exosomal miR-379-5p by macrophages increases M1 and macrophage release of galectin-3, a response which inhibits GC proliferation in antral but not preantral follicles. Androgen induces GC exosomal miR379 release from preantral but not antral follicles. The uptake of GC-derived exosomal miR379 suppresses PDK1 in macrophages, a cellular mechanism shifting macrophage polarization towards M1. M1 macrophage secretes higher level of galectin-3 in FF, a inflammatory cytokine that specifically suppressed GC proliferation in antral but not preantral follicles. (Created in BioRender.com ).

Article Snippet: Human recombinant galectin-3 (R & D systems; n = 3; 0.03 ng/mL) was added to non-PCOS granulosa cell cultures.

Techniques: Derivative Assay

Journal: Science Advances

Article Title: Proteolytic processing of galectin-3 by meprin metalloproteases is crucial for host-microbiome homeostasis

doi: 10.1126/sciadv.adf4055

Figure Lengend Snippet: ( A ) For MS-based HYTANE analysis for the enrichment of N-terminal peptides tissue lysates of the proximal and distal colon from wild-type, meprin α knockout ( mep1a −/− ), meprin β knockout ( mep1b −/− ), and meprin α and meprin β double knockout ( mep1a/b −/− ) mice were used ( n = 3). ( B ) Volcano plots showing all identified proteolytic events detected following HYTANE analysis of wild-type mice in comparison to mep1a −/− , mep1b −/− , or mep1a/b −/− mice. Gray lines represent threshold values (±0.58 for log 2 difference and P = 0.05). Proteolytic fragments of galectin-3 could be identified in all approaches, indicating cleavage events between Ser 112 and Gly 113 or Gly 114 and Tyr 115 . ( C ) Heatmaps of the top 25 highest (blue) and less (red) abundant proteolytic peptides from three biological replicates (BRs) (blue, wild-type > knockout; red, wild-type < knockout; sorted by log 2 difference). In all approaches, cleavage of galectin-3 is more abundant in wild-type mice in comparison to the protease knockout mice (highlighted with a blue box).

Article Snippet: For the cleavage of galectin-3 by the meprins, 20 μg of recombinant human galectin-3 (PeproTech) or 10 μg of murine recombinant galectin-3 (R&D Systems) was incubated with or without 5 nM human active recombinant meprin α and/or 5 nM human active recombinant meprin β ( ) in 20 mM Hepes at 37°C for 2 hours and then stored at −20°C until use.

Techniques: Knock-Out, Double Knockout

Journal: Science Advances

Article Title: Proteolytic processing of galectin-3 by meprin metalloproteases is crucial for host-microbiome homeostasis

doi: 10.1126/sciadv.adf4055

Figure Lengend Snippet: ( A ) Galectin-3 cleavage in colonic tissue from three biological replicates of wild-type, mep1a −/− , mep1b −/− , or mep1a/b −/− mice analyzed by Western blot using a specific galectin-3, meprin α, and meprin β antibody. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. Galectin-3 was only proteolytically processed in wild-type mice when meprin α/β heterodimers are present in the colon. Asterisk marks an unspecific band. ( B ) Scheme of the generation of the mep1b −/− ;Vil Cre ; Rosa26 mep1b-HA mice having an overall meprin β knockout with reexpressed meprin β–HA in enterocytes of the small and large intestines and Western blot analysis of colonic tissue of mep1b −/− and mep1b −/− ;Vil Cre ; Rosa26 mep1b-HA mice to visualize galectin-3 cleavage via a specific galectin-3 antibody. Meprin β was either detected using a specific meprin β antibody or an HA-tag antibody against meprin β C terminus. GAPDH served as a loading control. Asterisk marks an unspecific band. ( C ) Stimulated emission depletion (STED) microscopy of intestinal Swiss roles from the whole colon of mep1b −/− ;Vil Cre ; Rosa26 mep1b-HA mice, mep1a/b −/− mice, or lgals3 −/− mice. Tissues were stained against galectin-3 (red), meprin β (green), or villin-1 (red or green). For reexpressed HA-tagged meprin β, the HA-antibody was used. Nuclear staining was visualized using 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 5 μm.

Article Snippet: For the cleavage of galectin-3 by the meprins, 20 μg of recombinant human galectin-3 (PeproTech) or 10 μg of murine recombinant galectin-3 (R&D Systems) was incubated with or without 5 nM human active recombinant meprin α and/or 5 nM human active recombinant meprin β ( ) in 20 mM Hepes at 37°C for 2 hours and then stored at −20°C until use.

Techniques: Western Blot, Knock-Out, Microscopy, Staining

Journal: Science Advances

Article Title: Proteolytic processing of galectin-3 by meprin metalloproteases is crucial for host-microbiome homeostasis

doi: 10.1126/sciadv.adf4055

Figure Lengend Snippet: ( A ) Differentiation process of human colorectal adenocarcinoma Caco-2 cells seeded on a transwell for 21 days. ( B ) Scheme of an enterocyte (left) and cross-sectional view of the z-stacks from the cell monolayer of Caco-2 cells differentiated for 21 days (right) showing the localization of F-actin (green) and villin (red; bottom) on the apical side, ZO-1 (red; middle) between adjacent cells, and the nucleus (stained with DAPI; blue) on the basolateral side and E-cadherin (red; top) on the lateral membrane. ( C to E ) Relative mRNA expression of MEP1A (C), MEP1B (D), and LGALS3 (E) in undifferentiated (day 0; red) and in differentiated (day 21; blue) Caco-2 cells ( n = 9). Means ± SD. (*** P < 0.001; unpaired t test). ( F ) Endogenous proteolytic processing of galectin-3 in cells of undifferentiated (day 0) and differentiated (day 21) Caco-2 was analyzed via Western blot analysis using a specific galectin-3 antibody. GAPDH served as a loading control. ( G ) Treatment of differentiated Caco-2 cells (day 21) with 3 μM actinonin showed an abolished galectin-3 cleavage analyzed by immunoblotting using a galectin-3–specific antibody. ( H ) Genotyping of the human meprin α and meprin β genes ( MEP1A and MEP1B ) in differentiated Caco-2 [wild-type (WT), MEP1A −/− , and MEP1B −/− ] via polymerase chain reaction (PCR) using specific primers for validating CRISPR-Cas9 knockout of either meprin α or meprin β. ( I ) Western blot analysis investigating the cleavage of galectin-3 in cell lysates from differentiated (day 21) wild-type, MEP1A −/− , and MEP1B −/− Caco-2 cells generated with CRISPR-Cas9 genome editing.

Article Snippet: For the cleavage of galectin-3 by the meprins, 20 μg of recombinant human galectin-3 (PeproTech) or 10 μg of murine recombinant galectin-3 (R&D Systems) was incubated with or without 5 nM human active recombinant meprin α and/or 5 nM human active recombinant meprin β ( ) in 20 mM Hepes at 37°C for 2 hours and then stored at −20°C until use.

Techniques: Staining, Expressing, Western Blot, Polymerase Chain Reaction, CRISPR, Knock-Out, Generated

Journal: Science Advances

Article Title: Proteolytic processing of galectin-3 by meprin metalloproteases is crucial for host-microbiome homeostasis

doi: 10.1126/sciadv.adf4055

Figure Lengend Snippet: ( A ) For Caco-2 cell treatment with activity modulating compounds, the medium was changed to serum-free Dulbecco’s modified Eagles’s medium (DMEM) without phenol red in both compartments on day 17. For the meprin inhibition, 3 μM actinonin was added daily to the apical and basolateral compartment until day 21 (red; right). For the meprin activation, 5 nM RgpB, a purified protease from P. gingivalis , was added to both compartments on day 20 (blue; middle). As a control, untreated cells were analyzed (left). Cells were harvested on day 21 for HYTANE analysis. ( B to D ) Volcano plots showing all identified proteolytic events in differentiated Caco-2 cells detected by HYTANE analysis (untreated, activated, and inhibited). Gray lines represent threshold values (±0.58 for log 2 difference and P = 0.05). ( E ) Human galectin-3 sequence (amino acids 96 to 120) showing all identified cleavage sites using HYTANE analysis.

Article Snippet: For the cleavage of galectin-3 by the meprins, 20 μg of recombinant human galectin-3 (PeproTech) or 10 μg of murine recombinant galectin-3 (R&D Systems) was incubated with or without 5 nM human active recombinant meprin α and/or 5 nM human active recombinant meprin β ( ) in 20 mM Hepes at 37°C for 2 hours and then stored at −20°C until use.

Techniques: Activity Assay, Modification, Inhibition, Activation Assay, Purification, Sequencing

Journal: Science Advances

Article Title: Proteolytic processing of galectin-3 by meprin metalloproteases is crucial for host-microbiome homeostasis

doi: 10.1126/sciadv.adf4055

Figure Lengend Snippet: ( A ) Detection of galectin-3 cleavage fragments by Western blot analysis upon coexpression of galectin-3 (C-terminally HA-tagged) with meprin α and/or meprin β in wild-type HEK293T cells and HEK293T-deficient for ADAM10 and ADAM17 (ADAM10/17 −/− ). ( B and C ) Cell surface biotinylation assay of HEK293T ADAM10/17 −/− cells transfected with galectin-3, meprin α, and/or meprin β (C). Cell surface proteins were labeled with primary amine biotinylation, pulled down with magnetic streptavidin beads, and analyzed via Western blot analysis. Transferrin receptor (Tfr) and GAPDH served as a control. Cartoon of the proteolytic processing of galectin-3 by meprin α/β heterodimers at the membrane (B). ( D ) HEK293T ADAM10/17 −/− cells were transfected with galectin-3 and meprin α and/or meprin β. After cell lysis, co-IP was performed using an HA-tag antibody against galectin-3 C terminus. Lysate controls and immunoprecipitates were analyzed by Western blot.

Article Snippet: For the cleavage of galectin-3 by the meprins, 20 μg of recombinant human galectin-3 (PeproTech) or 10 μg of murine recombinant galectin-3 (R&D Systems) was incubated with or without 5 nM human active recombinant meprin α and/or 5 nM human active recombinant meprin β ( ) in 20 mM Hepes at 37°C for 2 hours and then stored at −20°C until use.

Techniques: Western Blot, Cell Surface Biotinylation Assay, Transfection, Labeling, Lysis, Co-Immunoprecipitation Assay

Journal: Science Advances

Article Title: Proteolytic processing of galectin-3 by meprin metalloproteases is crucial for host-microbiome homeostasis

doi: 10.1126/sciadv.adf4055

Figure Lengend Snippet: ( A ) Recombinant human (top) and murine (bottom) galectin-3 was incubated with recombinant active meprin α and/or meprin β and analyzed by SDS-PAGE and Coomassie brilliant blue staining. ( B ) Galectin-3 cleavage analyzed in transfected HEK293T ADAM10/17 -/- cells in the presence or absence of the meprin β sheddase ADAM17 detected by immunoblotting. ( C ) The cleavage site in human and murine galectin-3 was identified using LC-MS analysis of the gel bands in (A) as depicted in the structural model of galectin-3 (AlphaFold: AF-P16110-F1). Gray, CRD; blue, N-terminal tail. ( D ) Analysis of human recombinant galectin-3 with and without cleavage by meprins via native PAGE. ( E ) Scheme of the bacterial agglutination assay. ( F ) E. faecalis was incubated with recombinant galectin-3 alone or preincubated with active recombinant meprin α and/or meprin β, and bacterial agglutination was examined. As a negative control buffer, 20 mM Hepes, pH 7.2, or active recombinant meprins were applied without galectin-3. Middle panel original image. Right panel shows the area of nonagglutinated bacteria, which was calculated using ImageJ Fiji. All values were normalized to the control (Hepes), while the control corresponds to 0 and maximum agglutination to 1. ( G ) Relative bacterial agglutination of E. faecalis ( n = 4), P. aeruginosa ( n = 4), E. coli ( n = 4), and K. pneumoniae ( n = 4) incubated with galectin-3 or galectin-3 preincubated with meprin α and/or meprin β. Data are presented as means ± SD, and statistical analysis was assessed by two-way analysis of variance (ANOVA), followed by a Tukey posttest (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: For the cleavage of galectin-3 by the meprins, 20 μg of recombinant human galectin-3 (PeproTech) or 10 μg of murine recombinant galectin-3 (R&D Systems) was incubated with or without 5 nM human active recombinant meprin α and/or 5 nM human active recombinant meprin β ( ) in 20 mM Hepes at 37°C for 2 hours and then stored at −20°C until use.

Techniques: Recombinant, Incubation, SDS Page, Staining, Transfection, Western Blot, Liquid Chromatography with Mass Spectroscopy, Clear Native PAGE, Agglutination, Negative Control

Journal: Science Advances

Article Title: Proteolytic processing of galectin-3 by meprin metalloproteases is crucial for host-microbiome homeostasis

doi: 10.1126/sciadv.adf4055

Figure Lengend Snippet: ( A ) Immunoblot analysis of colonic lysates from wild-type, mep1a/b −/− , and lgals3 −/− mice. Asterisk marks an unspecific band. ( B ) Volcano plot showing N termini identified by HYTANE analysis of wild-type versus lgals3 −/− mice. Gray lines represent threshold values (±0.58 for log 2 difference and P = 0.05). ( C ) Scheme of a horizontal perfusion chamber for mucus growth rate analysis. ( D ) Mucus thickness was measured at 0, 15, and 30 min after mounting, and growth rate was calculated as the delta mucus thickness divided by time from wild-type and lgals3 −/− mice ( n = 3). ( E ) The mucus thickness was quantified in wild-type and lgals3 −/− mice by calculating the average tissue to beads distance in the penetrability assay ( n = 10). (D and E) Data are represented as means ± SD, and statistical analysis was assessed by an unpaired t test [ns (not significant)]. ( F ) Cross sections through the inner mucus layer of ex vivo wild-type and lgals3 −/− distal colon tissue as well as y / x -axis images in the inner mucus layer (red line) and on the mucus surface (blue line): DNA (green), U. Europaeus agglutinin I (UEA-1)–stained intercrypt mucus (red), wheat germ agglutinin (WGA)–stained (gray). Scale bar, 100 μm. ( G ) Shallow shotgun sequencing of the microbiome of wild-type, lgals3 −/− , and mep1a/b −/− from stool samples ( n = 6). ( H ) Normalized 16 S abundance analyzed by qRT-PCR from stool samples of lgals3 −/− and mep1a/b −/− mice in comparison to wild-type mice. Data are represented as means ± SD, and statistical analysis was assessed by two-way ANOVA, followed by a Tukey posttest (* P < 0.05).

Article Snippet: For the cleavage of galectin-3 by the meprins, 20 μg of recombinant human galectin-3 (PeproTech) or 10 μg of murine recombinant galectin-3 (R&D Systems) was incubated with or without 5 nM human active recombinant meprin α and/or 5 nM human active recombinant meprin β ( ) in 20 mM Hepes at 37°C for 2 hours and then stored at −20°C until use.

Techniques: Western Blot, Ex Vivo, Staining, Shotgun Sequencing, Quantitative RT-PCR

Journal: Science Advances

Article Title: Proteolytic processing of galectin-3 by meprin metalloproteases is crucial for host-microbiome homeostasis

doi: 10.1126/sciadv.adf4055

Figure Lengend Snippet: ( A ) Western blot analysis of colon tissue from conventionally housed (wild-type), germ-free, or wildling mice demonstrated an altered cleavage of galectin-3 in response to different microbiomes. ( B to D ) Densitometric analysis of cleaved galectin-3 (B), meprin α (C), and meprin β (D) calculated by ImageJ from three biological replicates of wild-type and germ-free mice and four biological replicates of wildling mice as shown in (A). Data are represented as means ± SD, and statistical analysis was assessed by one-way ANOVA, followed by a Tukey posttest (** P < 0.01; *** P < 0.001). ( E ) Analysis of proteolytic processing of galectin-3 and meprin expression in the colon of C. rodentium –infected mice (control, n = 4; C. rodentium , n = 6) 7 days after infection via immunoblotting. ( F to H ) Densitometric analysis of cleaved galectin-3 (F), meprin α (G), and meprin β (H) calculated by ImageJ from four or six biological replicates as shown in (E). Data are represented as means ± SD, and statistical analysis was assessed by an unpaired t test (ns, P > 0.05; * P < 0.05; ** P < 0.01). (B to D and F to H) The intensity of cleaved galectin-3, meprin α, and meprin β was determined relative to the normalized expression of GAPDH.

Article Snippet: For the cleavage of galectin-3 by the meprins, 20 μg of recombinant human galectin-3 (PeproTech) or 10 μg of murine recombinant galectin-3 (R&D Systems) was incubated with or without 5 nM human active recombinant meprin α and/or 5 nM human active recombinant meprin β ( ) in 20 mM Hepes at 37°C for 2 hours and then stored at −20°C until use.

Techniques: Western Blot, Expressing, Infection