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. 2023 Mar 2;222(4):e202206121. doi: 10.1083/jcb.202206121

Figure S5.

Figure S5.

A galectin-3–Lrp1 axis regulates RhoA activation and sealing zone formation in osteoclasts. (A) Lrp1, c-Src, Ctsk, and RhoA expression in wild-type osteoclasts differentiated and treated with exogenous 0.1 μM galectin-3 in the presence or absence of 50 nM RAP for 5 d at 37°C as assessed by Western blot. Results are representative of three independent experiments. (B and C) Phalloidin (red) and WGA-DAB staining of wild-type pre-osteoclasts cultured atop bone slices treated with exogenous 0.1 μM galectin-3 in the presence or absence of 50 nM RAP for 3 d at 37°C (B), and actin ring area per cell and resorption pit area quantified (C). Scale bar, upper 20 μm, lower 100 μm. Data are presented as mean ± SEM (n = 6 biological replicates). (D) Wild-type osteoclasts were cultured and treated with exogenous 0.1 μM galectin-3 in the presence or absence of 50 nM RAP 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). (E) TRAP (red), WGA-DAB, and phalloidin staining (red) of wild-type pre-osteoclasts cultured atop bone slices treated with vehicle, and DKO osteoclasts treated with vehicle or 50 nM RAP for 3 d at 37°C (n = 6 biological replicates). Scale bar, upper and middle 100 μm, lower 20 μm. (F) GALECTIN-3 expression and cleavage as assessed by Western blot in human osteoclasts differentiated from macrophages in the presence or absence of either an MMP9 function-blocking mAb (25 μg/ml) or MMP14 function-blocking antibody (DX-2400; 100 μg/ml). Results are representative of three independent experiments. (G and H) Calvaria isolated from wild-type and DKO mice were cultured in the presence or absence of 20 μg/ml GCS-100 for 5 d at 37°C, and supernatants or whole cell lysates collected for CTX-I ELISA (G) and TRAP activity (H), respectively. Data are presented as mean ± SEM (n = 6 biological replicates). (I and J) Actin ring area per cell (I) and phalloidin staining (magenta) with TRAP (green) immunofluorescence (J) are shown of wild-type and DKO calvaria explants cultured in the presence or absence of 20 μg/ml GCS-100 for 5 d at 37°C. Scale bar, 10 μm. Data are presented as mean ± SEM (n = 6 biological replicates). **P < 0.01. Statistical significance assessed using one-way ANOVA with Bonferroni correction. Source data are available for this figure: SourceData FS5.