Fig 2: No or limited effect of PGDHC on RANKL‐mediated osteoclastogenesis induced in CatB−/− mouse calvaria in vivo. (A, C) Live imaging and (B, D) quantification of male and female wild‐type (CatB+/+) and CatB knock‐out (CatB−/−) mice calvarias injected with RANKL or a combination of RANKL‐PGDHC, using in vivo bioluminescence imaging (Extreme II, Bruker) with a CatK specific fluorescent probe (Pro Sense 680, Perkin Elmer). (E, G) Histological staining of TRAP+cells on the sagittal suture/parietal bones of the experimental subjects detailed above. Haematoxylin was used as a nuclear counterstain. (F, H) The normalized number of TRAP+cells per mm2 represent the increase in TRAP positive cells identified in the male and female wild‐type (CatB+/+) and CatB knockout (CatB−/−) mice calvarias injected with RANKL or a combination of RANKL‐PGDHC

Fig 3: Effects of PGDHC on in vitro RANKL‐induced osteoclastogenesis using bone marrow derived macrophages (BMDMs) isolated from wild‐type (CatB+/+) and CatB‐knock out (CatB−/−) mice. Expression patterns of some osteoclast fusion and differentiation mRNA genes in RANKL‐stimulated BMDMs from male and female CatB+/+ and CatB−/− mice were measured by qRT‐PCR analysis and are presented as the fold change after normalization against the housekeeping gene GAPDH (A‐H). The number of osteoclasts differentiated from male and female CatB+/+ and CatB−/− RANKL‐stimulated BMDMs are presented as the total count of tartrate resistant acid phosphatase positive (TRAP+) multinucleated cells per treated well (I‐J). Representative images of TRAP+multinucleated cells from each experimental group (K). *p < 0.05; **p < 0.01; ***p < 0.001; (#p < 0.05; ##p < 0.01; ###p < 0.001) between CAtB+/+ and CatB−/− counterparts; Scale bar (200 μm) applies to all images

Fig 4: PGDHC promotes lysosomal membrane permeabilization in RANKL‐primed bone marrow‐derived macrophages (BMDMs) in vitro. Representative images of acridine orange staining (AO, green) and nuclei (Hoeschst; blue) (A) and fluorescence intensity quantification (B) in the cytoplasm of RANKL‐primed BMDMs after 48 h stimulation with PGDHC; fluorescence intensity was quantified as the ratio of AO/Hoechst. (C – D) Identification of galectin 3 (Lgals3) and lysosome associated membrane protein 1 (Lamp‐1) proteins associated with the lysosomal membrane permeabilization in the cell lysate of BMDMs after 48 h stimulation with RANKL and PGDHC. (E) Immunofluorescent staining of Lgals‐3 (red), Lamp‐1 (green), and nuclear counterstain (blue). Arrow heads point to areas of colocalization of Lgals‐3 and Lamp‐1. *p < 0.05; **p < 0.01; Scale bars, 50 μm

Fig 5: Loc14 impedes RANKL-induced osteoclast differentiation and function. (A) CCK-8 assay detected the viability of bone marrow-derived macrophages (BMMs) or osteoclast precursor cells (OPCs) (B) after treated with Loc14 in gradient concentration (0–20 μM) for 24h. (C) TRAP staining of osteoclasts under Loc14 treatment with gradient concentration (0, 0.625, 1.25, 2.5 and 5 μM), and (F) the number of TRAP-positive osteoclasts with different nuclei (n = 3, 6–9, and ≥10) were counted. Scale bar = 100 μm. (D) F-actin rings for osteoclasts with or without Loc14 treatment after osteoclast induction for 5 days. The F-actin rings (red) and nuclei (blue) of control and Loc14-treated group were stained with phalloidin-iflour 594 and DAPI, and visualized by a fluorescence microscope. Scale bar = 100 μm. (G) Quantification of osteoclasts with F-actin rings per group. (E) Acridine orange (AO) staining after osteoclast induction for 5 days with Loc14 treatment. Scale bar = 100 μm. (H) The ratio of fluorescence intensity (red to green) of AO staining was quantitatively measured using Image J software. Data were shown as means ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)