Sc 32790l

Normal human CD14⁺ monocytes (Lonza) were cultured in α-MEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin-streptomycin, and 20 ng/ml M-CSF for 6 d to expand the precursor population and differentiate into macrophages. Cells were then detached with Accutase (A1110501; Gibco), seeded on tissue-culture plastic dishes, and cultured with M-CSF (20 ng/ml) and RANKL (20 ng/ml) for 9 d to induce multinuclear osteoclast formation, or reseeded on bone slices for bone resorption assay (Raynaud-Messina et al., 2018 (link); Zhu et al., 2020 (link)). Human osteoclasts were cultured with isotype control IgG, MMP9 function-blocking monoclonal antibody (sc-12759L; Santa Cruz), and a function-blocking antibody anti-MMP14 (DX-2400) at 100 μg/ml (Ager et al., 2015 (link); Marshall et al., 2015 (link); Zhu et al., 2020 (link)), in the presence or absence of an anti–GALECTIN-3 blocking antibody (sc-32790L; Santa Cruz) or an anti-LRP1 blocking antibody (MA1-27198; Thermo Fisher Scientific) at 25 μg/ml (Chen et al., 2015 (link); Moxon et al., 2015 (link); Seguin et al., 2017 (link)). DX-2400 was provided by the Kadmon Corporation.

Calvariae from 5-d-old wild-type or DKO mice were isolated aseptically, cleaned, and cultured for 16 h at 37°C in 0.5 ml of BGJb medium (Life Technologies) containing 1 mg/ml BSA (fraction V; Sigma-Aldrich; Moxon et al., 2015 (link)). Half calvariae were transferred to fresh medium with 0.1 μM parathyroid hormone (H-4835.0005; Bachem) in the presence or absence of an anti–galectin-3 monoclonal antibody (sc-32790L; Santa Cruz) or an anti-Lrp1 monoclonal antibody (MA1-27198; Thermo Fisher Scientific), and cultured for an additional 5 d. Culture supernatant was collected for bone resorption marker CTX-I level detection using RatLaps CTX-I EIA kit (AC-06F1; Immunodiagnostic Systems). The half calvariae were either fixed for immunostaining with an anti-TRAP polyclonal antibody (sc-30833; Santa Cruz), an anti–galectin-3 monoclonal antibody (#125401; Biolegend; clone M3/38), or an anti-V-type proton pump-3 (Vpp3) polyclonal antibody (ab200839; Abcam) as describe above or snap-frozen for TRAP activity assay.

For bone resorption assays, BMDMs or pre-osteoclasts were seeded and cultured on bovine cortical bone slices (DT-1BON1000-96; Immunodiagnostic Systems) with 20 ng/ml M-CSF and 30 ng/ml RANKL (R&D Systems; Wu et al., 2017 (link); Zhang et al., 2018 (link); Zhu et al., 2020 (link)), in the presence or absence of galectin-3 (8259-GA; R&D Systems), galectin-3C (10110-GA; R&D Systems), GCS-100 (La Jolla Pharmaceutical), RAP (4480-LR; R&D Systems), an anti–galectin-3 blocking antibody (sc-32790L; Santa Cruz), or an anti-Lrp1 blocking antibody (MA1-27198; Thermo Fisher Scientific; Chen et al., 2015 (link); Demotte et al., 2010 (link); John et al., 2003 (link); Moxon et al., 2015 (link); Seguin et al., 2017 (link)). After the indicated culture period, bone samples were sonicated in PBS, stained with 20 μg/ml WGA-lectin (L3892; Sigma-Aldrich) for 45 min and then incubated with DAB tablets (D4418; Sigma-Aldrich) for 15 min. Image J software was used to quantify the resorbed area. The concentration of the CTX-I was measured using the CrossLaps for Culture CTX-I ELISA kit (AC-07F1; Immunodiagnostic Systems) according to the manufacturer’s instructions.