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

Figure 5.

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 phalloidin staining (red) of wild-type pre-osteoclasts cultured atop bone slices in the presence of exogenous 0.1 μM galectin-3 for 3 d at 37°C (C), with 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). (E) Wild-type osteoclasts were cultured and treated with exogenous 0.1 μM galectin-3 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). (F and G) Wild-type pre-osteoclasts were cultured atop bone slices and treated with exogenous 0.1 μM galectin-3C for 3 d at 37°C (John et al., 2003; Massa et al., 1993). The number of TRAP+ MNCs, resorption pit area, and actin ring area per cell were quantified (G) following TRAP, WGA-DAB, and phalloidin staining (red) (F), respectively. Scale bar, upper and middle 100 μm, lower 20 μm. Data are presented as mean ± SEM (n = 6 biological replicates). (H) Wild-type osteoclasts were cultured and treated with exogenous 0.1 μM galectin-3C 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). **P < 0.01. Statistical significance was assessed using unpaired two-sided Student’s t test. Source data are available for this figure: SourceData F5.