Fig 2: Osteoclast differentiation process of the palatal bone (PPP). (A) TRAP staining from E14.5 to E18.5 “a–e”, von Kossa staining from E14.5 to E18.5 “f–j”. Immunofluorescence staining expression patterns of CD31 from E14.5 to E18.5 “k–o”. (B–E) Immunofluorescence staining expression patterns of CD14, F4/80, CSF1R and RANKL during palatal osteoclastogenesis from E14.5 to E17.5. Black triangles represent TRAP-positive regions, and white triangles represent mineralizing and CD31-positive regions. Scale bar 500 µm.

Fig 3: Suppressive effect of calcitriol on RANKL expression in Th cells in an inflammatory environment. A, mRNA level of RANKL in Th cells (determined by qRT‐PCR) following incubations in various conditions (LPS group, LPS + DC group and LPS + DC + Cal group). B, Protein level of RANKL in Th cells (determined by Western blotting) following incubations in various conditions (LPS group, LPS + DC group and LPS + DC + Cal group; left panel) and semi‐quantitative analysis of the protein expression level (normalized to the level of actin) in terms of the relative grey density (right panel). C, Representative flow cytometry plots of IL‐17+/RANKL+ Th cells following incubations in various conditions (LPS group, LPS + DC group and LPS + DC + Cal group). D, Quantification of the proportion of IL‐17+/RANKL+ Th cells (assessed by flow cytometry). E, Representative immunofluorescence images of IL‐17+/RANKL+ Th cells (cell nucleus, blue fluorescence; IL‐17 protein, green fluorescence; RANKL protein, red fluorescence; scale bar: 100 μm). F, Quantification of the proportion of IL‐17+/RANKL+ Th cells (calculated from an immunofluorescence assay). The data are shown as the mean ± SD; *P < .05 and **P < .01 represent significant differences between the indicated columns

Fig 4: STING-P-TBK1 pathway blocking by siRNA alters inflammatory cytokines secretion and osteoclastogenic activity in an in vitro osteocyte model of irradiation. (A) The knockdown efficiency of STING was verified by western blot analyses. (B) After the STING-siRNA knockdown, the protein expression of P-TBK1 in MLO-Y4 cells was evaluated 3 days post-irradiation by western blot analysis. (C) Effect of STING-targeted siRNA intervention on the expression of bone resorption-related protein RANKL. (D) Effect of STING-targeted siRNA intervention on the mRNA expressions of inflammatory cytokines including IL-1α, IL-6, and NF-κB. (E) STING-targeted siRNA intervention on the paracrine regulation of osteoclastogenesis by IR-induced inflammatory cytokine from OCYs. The RAW264.7 cells were co-cultured with the conditioned medium (CM) of irradiated MLO-Y4 cells following STING-siRNA or NC siRNA intervention. The percentage of TRAP+ OCs was quantified (scale bar, 100 µm; magnification, ×100). Data are expressed as mean ± SD (* p < 0.05; ** p < 0.01; *** p < 0.001 vs. control), n = 3. NC siRNA: non-STING targeted siRNA interference; STING siRNA: STING targeted siRNA interference. The CM of MLO-Y4 cells was collected at 3 days after 0 Gy or 4 Gy irradiation with STING siRNA or NC siRNA intervention. CM-0 Gy-NC: non-irradiation with NC siRNA intervention; CM-4 Gy-NC: 4 Gy γ-radiation with NC siRNA intervention; CM-0 Gy-SI: non-irradiation with STING-siRNA intervention; CM-4 Gy-SI: 4 Gy γ-radiation with STING-siRNA intervention.

Fig 5: Introduction of ameloblastoma tumour mass to the 3D bone stroma prior to day 9 completely inhibits bone nodule formation. (a) Introduction of AM-1 and AM-3 tumour masses to 3D bone at day 6 inhibits bone nodule formation. Images taken at day 6, 9 and 21, 4 × Magnification, scale bar = 100 μm. ALPL expression. TNFSF11 (RANKL) Expression. (b) Introduction of AM-1 and AM-3 tumour masses to 3D bone at day 9 restricts bone formation by limiting bone nodule number and bone nodule surface area. (c) RT2 Profiler PCR Array was conducted to screen osteogenesis gene of osteoblasts in the 3D bone stroma model and in AM-3 tumour mass introduced 3D bone stroma model at day 8. The AM-3 tumour mass was introduced at day 6 of 3D bone stroma model. Volcano plot shows under-expressed, unchanged and over-expressed genes. The table represents > 3.5-fold under-expressed gene. Horizontal line p-value threshold (0.05). One-Way ANOVA, Dunnet’s Post Hoc; p-values 0.05 = *, 0.005 = **, 0.0005 = *** and 0.00005 = ****.