Normal human cd14 monocytes

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 days to expand the precursor population and differentiate into macrophages. Cells were then detached with Accutase (Gibco) and seeded on tissue culture plastic dishes or bone slices and cultured with M‐CSF (20 ng/ml) and RANKL (20 ng/ml) for 9 days to induce multinuclear osteoclast formation and bone resorption as previously described (Raynaud‐Messina et al, 2018 (link); Zhu et al, 2020 (link)). For RNA interference experiments, primary human macrophages were transfected with individual siRNA oligonucleotides using GenMute siRNA Transfection Reagent (SignaGen Laboratories) following the manufacturer's instructions (Aucher et al, 2013 (link)), which were then induced into human osteoclasts with M‐CSF and RANKL. Two independent siRNA specifically targeting ZEB1 (109652 and 109653, Thermo Fisher Scientific), two independent siRNA targeting CKMT1 (s3097 and s3099, Thermo Fisher Scientific), and the corresponding non‐targeting control siRNA (sc‐36869, Santa Cruz) were used. Expression of the siRNA knockdown efficiency was confirmed by Western blot.

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.