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

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© 2023 Zhu et al.

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Figure S3. — Active RhoA rescues defects in sealing zone formation and bone resorption in DKO osteoclasts. (A) Relative expression levels of small GTPase-mediated signal transduction-related transcripts retrieved from DAVID GO categories were assessed in DKO osteoclasts as compared with wild-type osteoclasts (normalized to 1; n = 3). (B) The differentially expressed transcripts of cytoskeleton-associated pathways in DKO osteoclasts as compared with wild-type osteoclasts are presented (n = 3; color bar, relative expression value). (C) Rac1 activity of wild-type and DKO osteoclasts cultured on plastic 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). (D) EV-transduced wild-type BMDMS, and EV- or ca-RhoA–transduced DKO BMDMs were differentiated into osteoclasts on plastic, and RhoA activity determined upon activation 20 ng/ml M-CSF and 30 ng/ml RANKL for 15 min. Data are presented as mean ± SEM (n = 6 biological replicates). (E) EV-transduced wild-type pre-osteoclasts, and EV- or ca-RhoA–transduced DKO pre-osteoclasts were cultured atop bone slices for 3 d and stained with phalloidin (red). Osteoclasts were removed and resorption pits visualized by WGA-DAB staining (n = 6 biological replicates). Scale bar, upper 20 μm, lower 100 μm. (F and G) Immunofluorescence staining of a femur section from a wild-type mouse with anti-galectin-3 (#125401; Biolegend; clone M3/38; F; green), anti-Mmp9, anti-Mmp14 antibodies (G; cyan), or corresponding isotype controls. BM cavity, bone marrow cavity. Scale bar, 30 μm. Results are representative of three independent experiments. (H) Galectin-3 expression and cleavage as assessed by Western blot in osteoclasts generated from wild-type, Mmp9^−/−, Mmp14^ΔM/ΔM, or DKO mice. Results are representative of three independent experiments. **P < 0.01. Statistical significance was assessed using unpaired two-sided Student’s t test (C) and one-way ANOVA with Bonferroni correction (D). Source data are available for this figure: SourceData FS3.