Journal: Molecular Biology Reports

Article Title: Human Coxsackie- and adenovirus receptor is a putative target of neutrophil elastase-mediated shedding

doi: 10.1007/s11033-022-07153-2

Figure Lengend Snippet: Human CAR recombinant extracellular domain (rhECD) is cleaved by serine proteases [neutrophil elastase (NE), cathepsin G (CG), proteinase 3 (PR3)] and matrix metalloproteinase 3 (MMP-3), but not MMP-1. rhECD was treated with proteases at a concentration of 100 ng/µl overnight at 37 °C and visualised with an anti-CAR N-terminus antibody (abcam; Cat.-No. ab189216) in Western blot. As negative control, protease reconstitution buffer was added instead of the protease. Distinct cleavage pattern with smaller products are visible

Article Snippet: Human proteinase 3 (PR3) from neutrophils (Enzo; Cat.-No. BML-SE498) was resuspended in deionised water.

Techniques: Recombinant, Concentration Assay, Western Blot, Negative Control

Journal: Molecular Biology Reports

Article Title: Human Coxsackie- and adenovirus receptor is a putative target of neutrophil elastase-mediated shedding

doi: 10.1007/s11033-022-07153-2

Figure Lengend Snippet: Recombinant human CAR extracellular domain (rhECD) is cleaved by serine proteases [proteinase 3 (PR3), cathepsin G (CG), neutrophil elastase (NE)] in a time- and dose-dependent manner. For time series (top panel for PR3 and CG and bottom panel left for NE), rhECD was treated with proteases (100 ng/µl) for different periods of time [5 min to overnight (o/n)] at 37 °C. As negative control, protease reconstitution buffer was added instead of the protease. For NE concentration series (bottom panel right), rhECD was treated with NE overnight at concentrations ranging from 0 to 100 ng/µl. Western blots were probed with anti-CAR N-terminus antibody (abcam; Cat.-No. ab189216), stripped, and reprobed with anti-C-terminal His tag antibody (abcam; Cat.-No. ab1187). PR3 and CG digests result in two cleavage products that include the N-terminal part of the protein and are not detected by the anti-C-terminal His tag antibody. However, reaction takes at least two hours and is not complete after overnight incubation. NE treatment results in an intermediate product (about 22 kDa) and an end product (about 17 kDa). Intermediate and end products are also observed, when NE is added in lower concentrations overnight. Those cleavage products comprise the N-terminal part of the protein and are not detected by the anti-C-terminal His tag antibody. A counter product (about 9 kDa) of the intermediate product is detected by anti-C-terminal His tag antibody at lower NE concentrations

Article Snippet: Human proteinase 3 (PR3) from neutrophils (Enzo; Cat.-No. BML-SE498) was resuspended in deionised water.

Techniques: Recombinant, Negative Control, Concentration Assay, Western Blot, Incubation

Journal: Scientific Reports

Article Title: Spindle pole cohesion requires glycosylation-mediated localization of NuMA

doi: 10.1038/s41598-017-01614-6

Figure Lengend Snippet: Galectin-3 loss leads to severe tissue disorganization during renal recovery. ( a ) Scheme showing the nephronic reduction procedure. ( b–c ) Histological analyses of sham-operated ( Control ) ( b ) and Nx ( c ) wt and gal3 −/− kidneys 3 months after surgery. Scale bars, 1 mm; inserts, 100 µm. ( d ) Statistical analysis of the ratio kidney weight over mouse weight in control and Nx, wt and gal3 −/− kidneys. ( e ) Statistical analysis of tubular dilatations. ( f ) Statistical analysis of the amount of serum creatinine in control and Nx wt and gal3 −/− kidneys. ( g ) Statistical analysis of the proliferative index in control and Nx wt and gal3 −/− kidneys. ( h ) Confocal microscopy analysis and 3D rendering of primary cilia ( white arrow arrowhead ) immunostained with monoclonal anti-acetylated α-tubulin antibody ( white ) in Nx wt and Nx gal3 −/− kidneys. Scale bars, 10 µm. ( i) Statistical analysis of the primary cilium length in control and Nx kidneys in wt and gal3 −/− mice. ( j ) Confocal microscopy analysis of the distribution of pericentrin ( white ) in Nx wt and Nx gal3 −/− kidneys. N ( Nx wt ) = 6 mice, N ( Nx gal3 −/−) = 6. ( k ) Confocal microscopy analysis of mitoses with PH3 immunostaining ( yellow ) in Nx kidneys. Arrowheads , irregular metaphasic plate; arrows , lagging chromosomes. ( l ) Statistical analysis of the percentage of cells with unconventional metaphasic plate morphology. In the Fig. 1, data are means ± SEM. Renal epithelium was demarcated with a dotted white line. Nuclei were detected by Hoechst 33342 staining ( blue ). Detailed information about quantifications is available in Supplementary Information.

Article Snippet: For rescue experiments, Galectin-3 or NuMA depletion were performed by transfecting Galectin-3 CRISPR/Cas9 Knockout or NuMA CRISPR/Cas9 Knockout plasmids purchased from Santa Cruz (Dallas, Texas, USA).

Techniques: Confocal Microscopy, Immunostaining, Staining

Journal: Scientific Reports

Article Title: Spindle pole cohesion requires glycosylation-mediated localization of NuMA

doi: 10.1038/s41598-017-01614-6

Figure Lengend Snippet: Galectin-3 is required for correct mitoses in epithelial cell cultures. ( a ) Confocal microscopy analysis of mitotic poles after α-tubulin immunostaining and actin detection in Hela cells. Scale bars, 5 µm. ( b ) Statistical analysis of the number of multipolar mitosis. n ( Control siRNA ) = 1406 cells, n ( Galectin-3 siRNA #1 ) = 1291, n ( Galectin-3 siRNA #2 ) = 1158. Data are means ± SEM. ( c ) Confocal microscopy analysis of centrosomal ( arrowheads ) and acentrosomal ( stars ) mitotic spindles. Cells were immunostained for α-tubulin ( green ) and centrin-3 ( red ). Scale bars, 5 µm. ( d ) Statistical analysis of the number of centrosomal or acentrosomal mitotic spindles. n ( Control siRNA ) = 48 cells; n ( Galectin-3 siRNA #1 ) = 55; n ( Galectin-3 siRNA #2 ) = 53. Data are means ± SEM. ( e ) Fluorescence microscopy analysis of ninein in control and Galectin-3 siRNA cells. Nuclei were detected by Hoechst 33342 staining ( blue ). A white dotted line specifies the area which has been used for ninein intensity quantification in ( f) . Scale bars, 5 µm. ( f ) Statistical analysis of the intensity of ninein fluorescence over α-tubulin fluorescence intensity along the mitotic spindle in control ( Control siRNA , black line ) and Galectin-3-depleted ( Galectin-3 siRNA#1 , red line and Galectin-3 siRNA#2, orange line ) metaphase Hela cells. n ( Control siRNA ) = 19 cells; n ( Galectin-3 siRNA#1 ) = 19 cells; n ( Galectin-3 siRNA#2 ) = 13 cells. ( g ) Fluorescence microscopy analysis of γ-tubulin in control and Galectin-3 siRNA cells. A white dotted line specifies the area which has been used for γ-tubulin intensity quantification in ( h) . Scale bars, 5 µm. ( h ) Statistical analysis of the intensity of γ-tubulin fluorescence over α-tubulin fluorescence intensity along the mitotic spindle in control ( black line ) and Galectin-3-depleted ( red and orange lines ) metaphase Hela cells. n ( Control siRNA ) = 26 cells; n ( Galectin-3 siRNA#1 ) = 19; n ( Galectin-3 siRNA#2 ) = 21. ( i) Fluorescence microscopy analysis of α-tubulin and γ-tubulin ( in pseudocolor spectrum) at spindle pole in bipolar mitosis. Images are mean projection of xz spindle centered through DNA staining. n ( Control siRNA ) = 32 cells; n ( Galectin-3 siRNA#1 ) = 22; n ( Galectin-3 siRNA#2 ) = 18. Detailed information about quantifications is available in Supplementary Information.

Article Snippet: For rescue experiments, Galectin-3 or NuMA depletion were performed by transfecting Galectin-3 CRISPR/Cas9 Knockout or NuMA CRISPR/Cas9 Knockout plasmids purchased from Santa Cruz (Dallas, Texas, USA).

Techniques: Confocal Microscopy, Immunostaining, Fluorescence, Microscopy, Staining

Journal: Scientific Reports

Article Title: Spindle pole cohesion requires glycosylation-mediated localization of NuMA

doi: 10.1038/s41598-017-01614-6

Figure Lengend Snippet: Galectin-3 associates with spindle poles. ( a,b ) Epifluorescence microscopy analysis of Galectin-3 distribution during mitosis. Hela cells were immunostained for Galectin-3 ( red ) and either α-tubulin ( a ) or γ-tubulin ( b ) ( green ). Galectin-3 is located at the spindle poles in prophase and metaphase ( arrows ), and with cleavage furrow in cytokinesis ( arrows ). Nuclei were detected by Hoechst 33342 staining ( blue ). Scale bars, 5 µm. ( c – f ) 3D-SIM microscopy analysis of Galectin-3 localization at spindle poles. Hela cells were immunostained for Galectin-3 ( red ) and either α-tubulin ( c,d ) or γ-tubulin ( e,f ) ( green ). Longitudinal ( c and e ) and top ( d and f ) spindle views are shown and close-ups on spindle pole top views are presented in inserts. Nuclei were detected by Hoechst 33342 staining ( blue ). Galectin-3 is associated with (−) end MTs neighbouring γ-TURC complexes at spindle poles ( arrows ). Scale bars, 5 µm.

Article Snippet: For rescue experiments, Galectin-3 or NuMA depletion were performed by transfecting Galectin-3 CRISPR/Cas9 Knockout or NuMA CRISPR/Cas9 Knockout plasmids purchased from Santa Cruz (Dallas, Texas, USA).

Techniques: Epifluorescence Microscopy, Staining, Microscopy

Journal: Scientific Reports

Article Title: Spindle pole cohesion requires glycosylation-mediated localization of NuMA

doi: 10.1038/s41598-017-01614-6

Figure Lengend Snippet: Galectin-3 and NuMA interaction relies on Galectin-3 lectinic activity. ( a ) Confocal microscopy analysis of endogenous Galectin-3 and transfected NuMA-GFP, and vice versa . Scale bars, 5 µm. ( b ) Western blot detection of NuMA ( Top blot, black arrowhead ) or Galectin-3 ( Bottom blot, black arrowhead ) after immunoprecipitation from mitotic extracts. Input, 10% of total cell lysate. ( c ) Western blot detection of NuMA ( Top blot, black arrowhead ) and Galectin-3 ( Bottom blot, black arrowhead ) after immunoprecipitation of NuMA or Galectin-3 in presence of Galectin-3-competitive sugars. Input, 10% of total cell lysate. ( d ) Confocal microscopy analysis of NuMA and α-tubulin in control and Galectin-3-depleted cells using CRISPR-Cas9 strategy, after transfection of Galectin-3-GFP, Galectin-3-R186S-GFP or Galectin-3-G182A-GFP. Scale bars, 5 µm. ( e) Statistical analysis of the number of multipolar cells after transfection of Galectin-3-GFP constructs in control or Galectin-3–depleted cells using CRISPR-Cas9 strategy. ( f ) Statistical analysis of the intensity of Galectin-3-GFP constructs over cytosol. ( g ) Western blot detection of Galectin-3 ( Top blot ) and NuMA ( Bottom blot ) after the immunoprecipitation of GFP from mitotic extracts of Galectin-3-depleted cells using CRISPR-Cas9 strategy, and transfected with Galectin-3-GFP constructs. Input, 10% of total cell lysate. ( h ) Fluorescence microscopy analysis of NuMA distribution in metaphase cells after DMSO or glycosylation inhibitor treatments. Scale bars, 5 µm. ( i ) Statistical analysis of the volume occupied by NuMA at spindle poles. ( j ) Fluorescence microscopy analysis of Galectin-3 distribution in metaphase cells after DMSO or glycosylation inhibitor treatments. Scale bars, 5 µm. ( k ) Statistical analysis of the intensity of Galectin-3 fluorescence at spindle poles over cytoplasmic Galectin-3 fluorescence intensity. ( l ) Western blot detection of NuMA ( Top blot ) and Galectin-3 ( Bottom blot, black arrowhead ) after the immunoprecipitation of NuMA from mitotic extracts, treated with DMSO or Ac-5SGlcNAc. Input, 10% of total cell lysate. Detailed information about quantifications is available in Supplementary Information.

Article Snippet: For rescue experiments, Galectin-3 or NuMA depletion were performed by transfecting Galectin-3 CRISPR/Cas9 Knockout or NuMA CRISPR/Cas9 Knockout plasmids purchased from Santa Cruz (Dallas, Texas, USA).

Techniques: Activity Assay, Confocal Microscopy, Transfection, Western Blot, Immunoprecipitation, CRISPR, Construct, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Spindle pole cohesion requires glycosylation-mediated localization of NuMA

doi: 10.1038/s41598-017-01614-6

Figure Lengend Snippet: Galectin-3 and NuMA interaction relies on O -GlcNAc modification of NuMA. ( a ) Schemes showing NuMA functional domains along its amino acid sequence: in light blue the N-terminal globular domain, in green the central coiled-coil domain and in dark blue the C-terminal globular domain (based on UniprotKB_Q14980). ( b ) Schemes showing the point mutations generated at NuMA O -GlcNAc glycosylation site. ( c ) Western blot analysis of NuMA ( Bottom blot ) and O-GlcNAc motifs ( Top blot ) after the immunoprecipitation of GFP from mitotic extracts of NuMA-depleted cells using CRISPR-Cas9 strategy, and transfected with NuMA-GFP constructs. ( d ) Confocal microscopy analysis of α-tubulin and γ-tubulin in control and NuMA-depleted cells using CRISPR-Cas9 strategy, after transfection of NuMA-3-GFP, NuMA-S1844A-GFP or NuMA-S1844T-GFP plasmids. Scale bars, 5 µm. ( e ) Statistical analyses of the number of multipolar cells after transfection of NuMA-GFP constructs in control or NuMA-depleted cells using CRISPR-Cas9 strategy. ( f ) Confocal microscopy analysis of mutated forms of NuMA and Galectin-3 in metaphase cells. Scale bars, 5 µm. Nuclei were detected by Hoechst 33342 staining ( blue ). ( g ) Statistical analysis of the Galectin-3 intensity over NuMA intensity after transfection of NuMA-GFP constructs in NuMA-depleted cells using CRISPR-Cas9 strategy. ( h ) Western blot analysis of NuMA ( Top blot ) and Galectin-3 ( Bottom blot ) after the immunoprecipitation of GFP from mitotic extracts of NuMA CRISPR KO cells transfected with NuMA-GFP constructs. Input, 10% of total cell lysate.

Article Snippet: For rescue experiments, Galectin-3 or NuMA depletion were performed by transfecting Galectin-3 CRISPR/Cas9 Knockout or NuMA CRISPR/Cas9 Knockout plasmids purchased from Santa Cruz (Dallas, Texas, USA).

Techniques: Modification, Functional Assay, Sequencing, Generated, Western Blot, Immunoprecipitation, CRISPR, Transfection, Construct, Confocal Microscopy, Staining

Journal: Scientific Reports

Article Title: Spindle pole cohesion requires glycosylation-mediated localization of NuMA

doi: 10.1038/s41598-017-01614-6

Figure Lengend Snippet: Galectin-3 acts in the organisation of NuMA at the spindle pole. ( a ) 3D-SIM microscopy analysis of α-tubulin ( green ) and Galectin-3 ( red ) distribution in control ( Control siRNA , upper panel ) and NuMA-silenced ( NuMA siRNA , lower panel ) Hela cells. Deprivation of NuMA leads to the loss of Galectin-3 recruitment at spindle poles and perturbation of mitotic spindles. Scale bars, 5 µm. ( b ) 3D-SIM microscopy analysis of α-tubulin ( green ) and NuMA ( red ) distribution in control ( Control siRNA , upper panel ) and Galectin-3 silenced ( Galectin-3 siRNA , lower panel ) Hela cells. NuMA is no longer focused at spindle poles and instead spreads along aberrant mitotic spindles ( arrowheads ) in the absence of Galectin-3. In addition, a cortical NuMA signal appears in Galectin-3 siRNA cells ( stars ). Scale bars, 5 µm. ( c ) Statistical analysis of the intensity of NuMA fluorescence over α-tubulin fluorescence intensity along the mitotic spindle in control ( Control siRNA , black line ) and Galectin-3-depleted ( Galectin-3 siRNA#1 , red line and Galectin-3 siRNA#2, orange line ) metaphase Hela cells. n ( Control siRNA ) = 24 cells; n ( Galectin-3 siRNA#1 ) = 24 cells; n ( Galectin-3 siRNA#2 ) = 24 cells. ( d ) Z-projection of spindle poles immunostained for NuMA in control ( Control siRNA , upper panel ) and Galectin-3-depleted ( Galectin-3 siRNA, lower panel ) metaphase Hela cells. ( e ) Statistical analysis of the volume occupied by NuMA at the spindle pole in control ( Control siRNA, white bar ) and Galectin-3-depleted ( Galectin-3 siRNA#1 , grey bar and Galectin-3 siRNA#2, black bar ) metaphase Hela cells. ( f ) Spinning-disc videomicroscopy analysis of NuMA-GFP localization in control ( Control siRNA , upper panels ) and Galectin-3-silenced ( Galectin-3 siRNA , lower panels ) metaphase Hela cells. Middle views going through the spindle poles ( yellow arrowheads ) are shown. Time frames from 0 to 4 min are presented.

Article Snippet: For rescue experiments, Galectin-3 or NuMA depletion were performed by transfecting Galectin-3 CRISPR/Cas9 Knockout or NuMA CRISPR/Cas9 Knockout plasmids purchased from Santa Cruz (Dallas, Texas, USA).

Techniques: Microscopy, Fluorescence

Journal: Scientific Reports

Article Title: Spindle pole cohesion requires glycosylation-mediated localization of NuMA

doi: 10.1038/s41598-017-01614-6

Figure Lengend Snippet: Galectin-3 participates in spindle pole cohesion and pericentrosomal matrix distribution. ( a ) 3D-SIM microscopy analysis of α-tubulin ( green ) and NuMA ( red ) after nocodazole treatment, wash out and MT regrowth for 0, 1, 5 or 20 min in control ( Control siRNA , left panels ) or Galectin-3-deprived ( Galectin-3 siRNA , right panels ). Nuclei were detected by Hoechst 33342 staining ( blue ). Scale bars, 5 µm. ( b ) Statistical analysis of metaphase morphology during MT regrowth at different time point (from left to right, separated by a doted-line, 1, 5 and 20 min after nocodazole washout/MT regrowth) in control and Galectin-3-depleted HeLa cells. Metaphase morphology has been set in four different categories; no MT ( white ): no MT appeared to be grown; MT foci ( light grey ): MT are organized in bouquet originating from a single γ-tubulin/NuMA foci; 2 spindles ( dark grey ): metaphase presented a classical bipolar organization; > 2 spindles ( black ): metaphase presented a multipolar profile. Data are means ± SEM. ( c ) Scheme showing the predicted mechanism of action of Galectin-3 in the stabilization of NuMA complexes and spindle pole cohesion in metaphase epithelial cells. As previously proposed , NuMA oligomerizes through its C-terminal globular domain ( dark blue ) to form radial structures that would interact altogether to form higher structure. NuMA interacts directly with MTs and dynein through its C-terminal globular domain ( dark blue ) and N-terminal region ( light blue ), respectively. The glycosylation ( purple ) is present on the C-terminal globular domain ( dark blue ). As Galectin-3 would recognize the glycosylation on the C-terminal globular domain, we hypothesize that it would facilitate the oligomerization of NuMA. This will contribute to a correct organization of spindle pole ( left panel ). We hypothesize that the absence of Galectin-3 might disturb NuMA oligomerized structures, affecting the structure of the spindle pole ( right panel ). Moreover, perturbation of the oligomerization of NuMA might lead to an increase of NuMA shuttling to the cortex through dynein-based transport and LGN interaction at plasma membrane ( question mark, right panel ). Detailed information about quantifications is available in Supplementary Information.

Article Snippet: For rescue experiments, Galectin-3 or NuMA depletion were performed by transfecting Galectin-3 CRISPR/Cas9 Knockout or NuMA CRISPR/Cas9 Knockout plasmids purchased from Santa Cruz (Dallas, Texas, USA).

Techniques: Microscopy, Staining