Fig 2: Immunofluorescence analysis of persistent expression of fusion-regulatory proteins in CD13-deficient multinucleated OC. Expression of the fusion regulatory proteins dynamin (a,d), DCST1 (b,e) and CD9 (c,f) is maintained in CD13KO multinucleated osteoclasts but not in WT cells (**). High levels of dynamin co-localize with actin and DCST1 in OCPs (*), imaged using Zeiss LSM 880 confocal fluorescence microscope and analyzed by Zeiss Zen 2.0 Pro blue edition software (https://www.zeiss.com/content/dam/Microscopy/Downloads/Pdf/FAQs/zen2-blue-edition_installation-guide.pdf). Scale bar; 10 µm. Data represents average of three independent experiments. N = 3/genotype. Dynamin, CD9, DCST1; green. Phalloidin; red. Magnification ×63 oil.

Fig 3: Substrate stiffness regulates the expression profile of osteoclast‐specific markers. (A–C) CLSM image of immunofluorescence‐stained cells (n = 3 independent experiments) showing changes in the expression levels of NFATc1, CTSK, and DCST1 (by red stain) on PDMS substrates of different rigidity. The osteoclasts were counterstained for F‐actin (phalloidin, green) and nuclei (DAPI, blue). (D) ImageJ quantification of the fluorescence intensity in (A–C) (n = 10 cells per group). Data are the mean ±SD. *p < .05, **p < .01. (E) Western blot assay of NFATc1, NF‐κB p65, CTSK, and DCST1 levels in cells cultured on PDMS substrates of different rigidity (n = 3 independent experiments). (F) Quantification of NFATc1, NF‐κB p65, CTSK, and DCST1 in (F). Data are the mean ±SD. Significant difference relative to group (1:5). *p < .05, **p < .01, **p < .001. (G) qPCR analysis of Traf6, Mmp9, Acp5, and Camk2a expression levels in stiff (1:5) and soft (1:45) groups (n = 3 independent experiments). *p < .05, ***p < .001

Fig 4: Immunoblot analysis of sustained expression of fusion-regulatory proteins in CD13-deficient OC over time. Expression of the fusion-promoting proteins dynamin, DC-STAMP (DCST1) and CD9 is aberrantly sustained in CD13KO but not in WT multinucleated osteoclasts (a,b) in presence of M-CSF and RANKL. A cropped image is presented, see Supplementary Fig. S7 for full-length blots and Supplementary Fig. S8 for cropped replicates. (c,d). CD13 expression is unaltered in BM-derived OCs in response to M-CSF and RANKL stimulation over time. Blots were imaged by ChemiDoc Imaging system version 3.0.1 (https://www.bio-rad.com/en-us/category/chemidoc-imaging-systems?ID=NINJ0Z15) (Biorad). A cropped image is presented, see Supplementary Fig. S9 for full-length blots and cropped replicates. Data represents average of two isolates. N = 3/genotype. **p < 0.01, *p < 0.05.

Fig 5: Immunofluorescence analysis of persistent expression of fusion-regulatory proteins in CD13-deficient multinucleated OC. Expression of the fusion regulatory proteins dynamin (a,d), DCST1 (b,e) and CD9 (c,f) is maintained in CD13KO multinucleated osteoclasts but not in WT cells (**). High levels of dynamin co-localize with actin and DCST1 in OCPs (*), imaged using Zeiss LSM 880 confocal fluorescence microscope and analyzed by Zeiss Zen 2.0 Pro blue edition software (https://www.zeiss.com/content/dam/Microscopy/Downloads/Pdf/FAQs/zen2-blue-edition_installation-guide.pdf). Scale bar; 10 µm. Data represents average of three independent experiments. N = 3/genotype. Dynamin, CD9, DCST1; green. Phalloidin; red. Magnification ×63 oil.