Fig. 2

Loss of Rufy4 impairs osteoclast function due to defective lysosome distribution. a–h BMMs from 6–8-week-old male Rufy4^+/+ and Rufy4^−/− mice were cultured with M-CSF and RANKL on bone slices. a, b Hematoxylin (a) or WGA (b) staining was conducted to visualize the resorption pit area and depth, respectively (n = 3). Scale bars, 250 μm (a) and 10 μm (b). c CTX-1 protein levels in the culture medium (n = 8). d Calcium levels in the culture medium (n = 6). e Immunoblot analysis of active and inactive CTSK levels in the cell lysates and supernatants (n = 3). Actin served as a loading control. Western blots were probed with the indicated antibodies. Band intensity was determined with ImageJ. f Schematic depiction of a mature osteoclast cultured on bone. g, h Immunofluorescence staining of CTSK (g) or Rab7 and LAMP2 (h) in osteoclasts at the bone surface level as described above in (f). Mean intensity of each protein per actin ring or the percentage of both Rab7⁺ and LAMP2⁺ in actin ring was quantified (n = 3). The dashed lines indicate the actin rings in osteoclasts. Scale bars, 10 μm. i, j BMMs from 6–8-week-old male Rufy4^+/+ and Rufy4^−/− mice were cultured with M-CSF and RANKL on a plastic surface. Flow cytometry of pHrodo green dextran (i) or lysosome enzyme activity (j) was conducted. Mean fluorescence intensity (MFI) of FITC was quantified (n = 5). k Representative electron microscopic images of Rufy4^+/+ and Rufy4^−/− osteoclasts cultured on bone slices. White asterisks and white arrows indicate mitochondria and autophagosomes, respectively. Scale bar, 1 μm. WGA wheat germ agglutinin, CTSK cathepsin K, Sup supernatants, RB ruffled border. The data are presented as mean ± SD. Two-tailed unpaired Student’s t test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.000 1; n.s. not significant