Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 1. Effect of MMP9 deficiency on embryonic kidney mat- uration. Representative photographs of E11.5 (A and B) and E12.5 (C and D) kidneys from MMP9/ (A and C) and MMP9/ mice (B and D) stained with calbindin D-28K to delineate ureteric bud (arrow). Please note the defect in ureteric bud elongation and branching in MMP9/ embryonic kidney. Bar: 250 m.

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques: Staining

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 2. Effect of MMP9 deficiency on mouse body weight, kidney weight, and nephron number. (A and B) Body weight of newborn (A) and adult (B) MMP9/ and MMP9/ kidneys. (C and D) Kidney weight of newborn (C) and adult (D) MMP9/ and MMP9/ kidneys. Kidney weight and body weight were similar at birth, whereas they were significantly decreased in adult MMP9/ mice. (E and F) Nephron number in newborn (E) and adult (F) MMP9/ and MMP9/ kidneys. Values are mean SEM. ***P 0.001 and **P 0.01 versus MMP9/ mice.

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques:

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 3. Effect of MMP9 deficiency on renal func- tion, kidney fibrosis, and kidney morphology. (A) Serum creatinine level of MMP9/ and MMP9/

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques:

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 4. Effect of MMP9 deficiency on ap- optosis ex vivo. (A and B) Representative photographs of E11.5 kidneys from MMP9/ (A) and MMP9/ mice (B) stained with calbindin D-28K to delineate ureteric bud in red and TUNEL method (apoptotic nuclei in green). (C) Percentage of apoptotic nuclei in E11.5 kidneys. Microphotographs were taken from 10 different MMP9/ and MMP9/ kidneys. (D) Concentration of ac- tive caspase-3 in E11.5 kidneys, n 50. Note that apoptosis assessed by different methods was increased in MMP9-deficient mesen- chyme. Values are mean SEM. ***P 0.001 versus MMP9/-injected mice. Bar: 250 m.

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques: Ex Vivo, Staining, TUNEL Assay, Concentration Assay, Injection

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 5. Effect of MMP9 deficiency on ap- optosis in vitro. (A and B) Representative pho- tographs of E11.5 kidneys from MMP9/ (A) and MMP9/ mice (B) grown for 48 h in culture. Ureteric bud is stained with calbindin D-28K (red) and apoptotic nuclei by the TUNEL method (green). (C) Percentage of ap- optotic nuclei in E11.5 kidneys grown for 48 h. (D) Genomic DNA extracted from E11.5 control (lane 1) and MMP9/ kidney (lane 2) grown for 48 h in culture. The migration pro- file in lane 2 is characteristic of apoptosis. Values are mean SEM. ***P 0.001 versus MMP9/ mice, n 20. Bars: (A and B) 250 and (E and F) 25 m.

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques: In Vitro, Staining, TUNEL Assay, Control, Migration

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 6. Effect of MMP9 deficiency on SCF (A and B) and c-kit (C and D) expression in E11.5 kidneys. (A) Representative SCF Western blot performed with 20 g of total proteins from MMP9/ (lane 1) and MMP9/ (lane 2) kidneys. (B) Quantitative analysis of five blots, using -actin as an internal control, showed a 50% increase of SCF protein expression in MMP9/ kidneys. (C) Representative Western blots detecting c-kit (lanes 1 and 2) and phospho c-kit (lanes 3 and 4) in 20 g total proteins extracts from MMP9/ (lanes 1 and 3) and MMP9/ (lanes 2 and 4) kidneys. (D) Phospho c-kit expression related to -actin expres- sion. Note that c-kit expression is not modified by MMP9 defi- ciency, whereas phospho c-kit expression is reduced by 60% in MMP9/ kidneys. Values are mean SEM. ***P 0.001 versus MMP9/ mice.

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques: Expressing, Western Blot, Control, Modification

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 7. Sensitivity of membrane SCF to MMP9 proteolysis in vitro. (A) Representative SCF Western blot performed with con- centrated medium obtained from 10 E11.5 MMP9/ (lane 1) or MMP9/ (lane 2) kidneys grown for 24 h. A band corresponding to the soluble form of SCF is observed in concentrated media from MMP9/ kidney only. (B) ELISA of sSCF in the culture medium of E11.5 MMP9/ (white bar) or MMP9/ (gray bar) kidneys grown for 24, 48, and 72 h. Values are mean SEM. ***P 0.001 versus MMP9/ mice, n 20.

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques: Membrane, In Vitro, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 8. Effect of sSCF on apoptosis in vitro. Representative photographs of E11.5 kidneys from MMP9/ (A through F) and MMP9/ (G through L) kidneys stained with calbindin D-28K to delineate ureteric bud in red and the TUNEL method (apoptotic nuclei in green). Kidneys were grown for 48 h in the absence (A through C and G through I) or the presence (D through F and J through L) of physiologic concentration of sSCF in culture medium. sSCF in culture medium decreased the number of apoptotic cells and increased ureteric bud branching of MMP9/ (J through L versus G through I) and MMP9/ (D through F versus A through C) kidneys. Bar: 250 m.

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques: In Vitro, Staining, TUNEL Assay, Concentration Assay

Journal: Journal of the American Society of Nephrology

Article Title: MMP9 Limits Apoptosis and Stimulates Branching Morphogenesis During Kidney Development

doi: 10.1681/asn.2009030312

Figure Lengend Snippet: Figure 9. Effect of sSCF on E11.5 MMP9/ and MMP9/

Article Snippet: Finally, caspase-3 released from tissue was measured in culture media obtained from five sets of ten MMP9 / and ten MMP9 / E12.5 embryos grown for 2 h by an ELISA assay developed by R&D Systems (Quantikine human active caspase-3).

Techniques:

Journal: The Journal of cell biology

Article Title: Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

doi: 10.1083/jcb.202206121

Figure Lengend Snippet: Figure 1. Genome wide analysis of gene expression programs in Mmp9/Mmp14 DKO osteoclasts. (A) BMDMs were isolated from wild-type or DKO mice and cultured on plastic substrata with 20 ng/ml M-CSF and 30 ng/ml RANKL for 5 d, stained with TRAP, and the number of TRAP+ MNCs was determined. Scale

Article Snippet: Recombinant human GALECTIN-3 protein (A13506; Abclonal) was incubated with activated recombinant MMP9 (RP00103; Abclonal) by adding 5 mM p-aminophenylmercuric acetate (A9563; Sigma-Aldrich) at 37°C for 30 min in 50 mM Tris-HCl buffer containing 150 mM NaCl and 10 mM CaCl2 (pH 7.5).

Techniques: Genome Wide, Gene Expression, Isolation, Cell Culture, Staining

Journal: The Journal of cell biology

Article Title: Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

doi: 10.1083/jcb.202206121

Figure Lengend Snippet: Figure 2. Mmp9/Mmp14-dependent RhoA activation and cytoskeletal organization. (A) RhoA activity of wild-type and DKO osteoclasts cultured on plastic upon activation with 20 ng/ml M-CSF and 30 ng/ml RANKL for 15 min (n = 6 biological replicates). (B and C) After a 6-d culture atop bone slice,

Article Snippet: Recombinant human GALECTIN-3 protein (A13506; Abclonal) was incubated with activated recombinant MMP9 (RP00103; Abclonal) by adding 5 mM p-aminophenylmercuric acetate (A9563; Sigma-Aldrich) at 37°C for 30 min in 50 mM Tris-HCl buffer containing 150 mM NaCl and 10 mM CaCl2 (pH 7.5).

Techniques: Activation Assay, Activity Assay, Cell Culture

Journal: The Journal of cell biology

Article Title: Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

doi: 10.1083/jcb.202206121

Figure Lengend Snippet: Figure 3. Dual Mmp9/Mmp14 deletion does not affect Cthrc1 levels or galectin-3 expression in osteoclasts. (A) Cthrc1 (green) immunofluorescence stained with an anti-Cthrc1 polyclonal antibody (ab85739; Abcam) and F-actin (red) imaged following phalloidin staining of wild-type and DKO osteoclasts.

Article Snippet: Recombinant human GALECTIN-3 protein (A13506; Abclonal) was incubated with activated recombinant MMP9 (RP00103; Abclonal) by adding 5 mM p-aminophenylmercuric acetate (A9563; Sigma-Aldrich) at 37°C for 30 min in 50 mM Tris-HCl buffer containing 150 mM NaCl and 10 mM CaCl2 (pH 7.5).

Techniques: Expressing, Immunofluorescence, Staining

Journal: The Journal of cell biology

Article Title: Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

doi: 10.1083/jcb.202206121

Figure Lengend Snippet: Figure 4. Mmp9/Mmp14 regulates galectin-3 lattice remodeling in osteoclasts. (A and B) Galectin-3 expression and cleavage as assessed with anti–galectin-3 monoclonal antibody (ab2785; Abcam; epitopes mapped against N-terminal region) by Western blot (A) and quantification (B) in BMDMs and osteoclasts (OC) generated from wild-type or DKO mice. Data are presented as mean ± SEM (n = 3 biological replicates). (C and D) Galectin-3 (green) im- munofluorescence of non-permeabilized wild-type or DKO osteoclasts stained with an anti–galectin-3 monoclonal antibody (#125401; Biolegend; clone M3/38, epitopes mapped against N-terminal region; C), and surface galectin-3 level quantified (D). Scale bar, 20 μm. Data are presented as mean ± SEM (n = 3 biological replicates with two technical replicates each). (E) Following cell surface biotinylation and capture with streptavidin magnetic beads, Mmp9, Mmp14, galectin-3, and β3 integrin expression in the membrane fraction of wild-type and DKO osteoclasts as assessed by Western blot. Results are representative of three independent experiments. (F) Measurements of surface galectin-3 in wild-type and DKO osteoclasts with eFluor 660–conjugated anti–galectin-3 monoclonal antibody (#50-5301-82; Thermo Fisher Scientific; clone M3/38, epitopes mapped within the N-terminal domain) by flow cytometry. Results are representative of three independent experiments. (G) Measurements of surface galectin-1 in wild-type and DKO osteoclasts by flow cytometry. Results are representative of three independent experiments. (H) DKO BMDMs were transduced with lentiviral vectors expressing full-length MMP9, an MMP9E/A mutant, or an empty control, and differentiated into osteoclasts. Cell lysates were collected for MMP9 and galectin-3 expression and cleavage as assessed by Western blot. Results are representative of three independent experiments. (I) DKO BMDMs were transduced with lentiviral vectors expressing full-length MMP14, MMP14E/A, or an empty control and differentiated into osteoclasts. Cell lysates were collected for MMP14 and galectin-3 expression and cleavage as assessed by Western blot. Results are representative of three independent experiments. **P < 0.01. Statistical significance was assessed using two-way ANOVA with Bonferroni cor- rection (B) and unpaired two-sided Student’s t test (D). Source data are available for this figure: SourceData F4.

Article Snippet: Recombinant human GALECTIN-3 protein (A13506; Abclonal) was incubated with activated recombinant MMP9 (RP00103; Abclonal) by adding 5 mM p-aminophenylmercuric acetate (A9563; Sigma-Aldrich) at 37°C for 30 min in 50 mM Tris-HCl buffer containing 150 mM NaCl and 10 mM CaCl2 (pH 7.5).

Techniques: Expressing, Western Blot, Generated, Staining, Magnetic Beads, Membrane, Flow Cytometry, Transduction, Mutagenesis, Control

Journal: The Journal of cell biology

Article Title: Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

doi: 10.1083/jcb.202206121

Figure Lengend Snippet: Figure 5. Exogenous galectin-3, but not galectin-3C, regulates osteoclast RhoA GTPase activation, sealing zone formation and bone resorptive activity. (A) Surface galectin-3 levels in wild-type osteoclasts exogenously treated with 0.1 μM galectin-3 or galectin-3C (corresponds to amino acids 108–250 containing the C-terminal carbohydrate recognition domain of galectin-3) for 30 min at 37°C (John et al., 2003; Massa et al., 1993), followed by staining with the CoraLite 488–conjugated galectin-3 polyclonal antibody (CL488-14979; Proteintech; epitopes mapped against full-length) as detected by flow cytometry. Results are representative of three independent experiments. (B) Mmp9, Mmp14, Ctsk, c-Src, and RhoA expression in wild-type osteoclasts treated with exogenous 0.1 μM galectin-3 as assessed by Western blot. Results are representative of three independent experiments. (C and D) TRAP (red), WGA-DAB, and

Article Snippet: Recombinant human GALECTIN-3 protein (A13506; Abclonal) was incubated with activated recombinant MMP9 (RP00103; Abclonal) by adding 5 mM p-aminophenylmercuric acetate (A9563; Sigma-Aldrich) at 37°C for 30 min in 50 mM Tris-HCl buffer containing 150 mM NaCl and 10 mM CaCl2 (pH 7.5).

Techniques: Activation Assay, Activity Assay, Staining, Flow Cytometry, Expressing, Western Blot

Journal: The Journal of cell biology

Article Title: Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

doi: 10.1083/jcb.202206121

Figure Lengend Snippet: Figure 6. Mmp9/Mmp14-dependent galectin-3 lattice remodeling controls osteoclast RhoA activation, sealing formation and bone resorptive ac- tivity. (A) Surface galectin-3 levels were determined in wild-type osteoclasts (OC) treated with control IgG, and DKO osteoclasts treated with control IgG or galectin-3 function-blocking mAb at 25 μg/ml for 2 h at 37°C, followed by staining with the CoraLite 488–conjugated galectin-3 full-length polyclonal antibody (CL488-14979; Proteintech) as detected by flow cytometry. Results are representative of three independent experiments. (B) Wild-type osteoclasts were treated with control IgG, and DKO osteoclasts were treated with control IgG or galectin-3 function-blocking mAb at 25 μg/ml for 2 h at 37°C, and RhoA activity determined upon activation with 20 ng/ml M-CSF and 30 ng/ml RANKL for 15 min. Data are presented as mean ± SEM (n = 6 biological replicates). (C and D) TRAP (red), WGA-DAB, and phalloidin staining (red) of wild-type pre-osteoclasts cultured atop bone slices treated with control IgG, and DKO pre-osteoclasts treated with control IgG or galectin-3 function-blocking mAb (Gal3 mAb; 25 μg/ml) for 3 d at 37°C (C), and the number of TRAP+ MNCs, resorption pit area, and actin ring area per cell quantified (D). Scale bar, upper and middle 100 μm, lower 20 μm. Data are presented as mean ± SEM (n = 6 biological replicates). *P < 0.05, **P < 0.01. Statistical significance was assessed using one-way ANOVA with Bonferroni correction.

Article Snippet: Recombinant human GALECTIN-3 protein (A13506; Abclonal) was incubated with activated recombinant MMP9 (RP00103; Abclonal) by adding 5 mM p-aminophenylmercuric acetate (A9563; Sigma-Aldrich) at 37°C for 30 min in 50 mM Tris-HCl buffer containing 150 mM NaCl and 10 mM CaCl2 (pH 7.5).

Techniques: Activation Assay, Control, Blocking Assay, Staining, Flow Cytometry, Activity Assay, Cell Culture

Journal: The Journal of cell biology

Article Title: Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

doi: 10.1083/jcb.202206121

Figure Lengend Snippet: Figure 7. Mmp9/Mmp14 co-dependent regulation of osteoclast function through the galectin-3–centric control of Lrp1 activation. (A–C) Pre- osteoclasts were incubated with either human GALECTIN-3 or GALECTIN-3-biotin (hGAL3-biotin; 1.5 µM) for 30 min at 4°C. Biotinylated GALECTIN-3 was precipitated with streptavidin magnetic beads, separated by SDS-PAGE (A), and analyzed by mass spectrometry (B). Hits were classified according to the peptides spectral matches and the number of peptides per protein. Red columns: the top hit of GALECTIN-3 binding partner. The numbering on the x axis relates to Table S3. Pie chart depicts GALECTIN-3 interactors based on the GO annotation “cellular component” in FunRich (C). (D) Lrp1 and galectin-3 ex- pression from the above streptavidin magnetic beads–precipitated lysates as assessed by Western blot. Results are representative of three independent experiments. (E) Protein lysates from wild-type osteoclasts were immunoprecipitated with the galectin-3 antibody, and Lrp1 and galectin-3 levels assessed by Western blot. Results are representative of three independent experiments. (F and G) Cell-free binding assay demonstrating direct binding between 0.1 and 3.2 μg GALECTIN-3 and 0.5 μg LRP1 for 4 h at 30°C (F), and the competitive inhibition of binding between GALECTIN-3 and LRP1 (1.6 versus 0.5 μg) by 50 mM sucrose, 50 mM lactose, or 10 mg/ml GCS-100 (G). Data are presented as mean ± SEM (n = 3 biological replicates). (H) Following cell surface biotinylation and capture with streptavidin magnetic beads, Lrp1 and β3 integrin expression in the membrane fraction of wild-type and DKO osteoclasts as assessed by Western blot. Results are representative of three independent experiments. **P < 0.01. Statistical significance was assessed using one-way ANOVA with Bonferroni correction. Source data are available for this figure: SourceData F7.

Article Snippet: Recombinant human GALECTIN-3 protein (A13506; Abclonal) was incubated with activated recombinant MMP9 (RP00103; Abclonal) by adding 5 mM p-aminophenylmercuric acetate (A9563; Sigma-Aldrich) at 37°C for 30 min in 50 mM Tris-HCl buffer containing 150 mM NaCl and 10 mM CaCl2 (pH 7.5).

Techniques: Control, Activation Assay, Incubation, Magnetic Beads, SDS Page, Mass Spectrometry, Binding Assay, Western Blot, Immunoprecipitation, Inhibition, Expressing, Membrane

Journal: The Journal of cell biology

Article Title: Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

doi: 10.1083/jcb.202206121

Figure Lengend Snippet: Figure 9. MMP9/MMP14–GALECTIN-3–LRP1 axis regulates human osteoclast activity. (A) GALECTIN-3 expression and cleavage as assessed with anti–galectin-3 monoclonal antibody (ab2785; Abcam; epitopes mapped against N-terminal region) by Western blot in human osteoclasts cultured in the

Article Snippet: Recombinant human GALECTIN-3 protein (A13506; Abclonal) was incubated with activated recombinant MMP9 (RP00103; Abclonal) by adding 5 mM p-aminophenylmercuric acetate (A9563; Sigma-Aldrich) at 37°C for 30 min in 50 mM Tris-HCl buffer containing 150 mM NaCl and 10 mM CaCl2 (pH 7.5).

Techniques: Activity Assay, Expressing, Western Blot, Cell Culture

Journal: The Journal of cell biology

Article Title: Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

doi: 10.1083/jcb.202206121

Figure Lengend Snippet: Figure 10. Mmp9/Mmp14–galectin-3–Lrp1 axis controls osteoclast activity in a calvaria explant model. (A and B) Calvaria isolated from wild-type and DKO mice were cultured in the presence or absence of either the galectin-3 or Lrp1 function-blocking mAbs (25 μg/ml) for 5 d at 37°C, and supernatants or

Article Snippet: Recombinant human GALECTIN-3 protein (A13506; Abclonal) was incubated with activated recombinant MMP9 (RP00103; Abclonal) by adding 5 mM p-aminophenylmercuric acetate (A9563; Sigma-Aldrich) at 37°C for 30 min in 50 mM Tris-HCl buffer containing 150 mM NaCl and 10 mM CaCl2 (pH 7.5).

Techniques: Activity Assay, Isolation, Cell Culture, Blocking Assay