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: Galectin-3 surface binding antagonist reverses functional defects in DKO osteoclasts. (A and B) Galectin-3 cleavage by Western blot using either an anti–galectin-3 full-length polyclonal antibody (Proteintech, 14979-1-AP; A) or an anti–galectin-3 monoclonal antibody (ab2785; Abcam; epitope mapped within the N-terminal region; B), following co-incubation with 3 μg recombinant human GALECTIN-3 and activated recombinant MMP9 at molar ratios from 1:50 to 1:5 (enzyme to substrate ratio) at 37°C for 30 min.

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 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: Galectin-3 surface binding antagonist reverses functional defects in DKO osteoclasts. (A and B) Galectin-3 cleavage by Western blot using either an anti–galectin-3 full-length polyclonal antibody (Proteintech, 14979-1-AP; A) or an anti–galectin-3 monoclonal antibody (ab2785; Abcam; epitope mapped within the N-terminal region; B), following co-incubation with 3 μg recombinant human GALECTIN-3 and activated recombinant MMP9 at molar ratios from 1:50 to 1:5 (enzyme to substrate ratio) at 37°C for 30 min.

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: Galectin-3 surface binding antagonist reverses functional defects in DKO osteoclasts. (A and B) Galectin-3 cleavage by Western blot using either an anti–galectin-3 full-length polyclonal antibody (Proteintech, 14979-1-AP; A) or an anti–galectin-3 monoclonal antibody (ab2785; Abcam; epitope mapped within the N-terminal region; B), following co-incubation with 3 μg recombinant human GALECTIN-3 and activated recombinant MMP9 at molar ratios from 1:50 to 1:5 (enzyme to substrate ratio) at 37°C for 30 min.

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