Fig 2: Effect of SIRT3 silencing on GAS promotion of osteogenic differentiation in LPS-stimulated hPDLSCs. (A) ALP expression in hPDLSCs was measured using ALP kit. (B) Alizarin Red staining images of hPDLSCs (magnification, x200). (C) Alizarin Red staining quantification. (D) Expression of ALP and Runx2 was detected by western blotting. (E) Expression of OCN and OPN was detected by western blotting. ***P<0.001 vs. control; ###P<0.001 vs. LPS; ?P<0.05, ??P<0.01 vs. GAS + LPS + shRNA-NC. ALP, alkaline phosphatase; GAS, gastrodin; hPDLSCs, human periodontal ligament stem cells; LPS, lipopolysaccharide; NC, negative control; OCN, osteocalcin; OD, optical density; OPN, osteopontin; Runx2, RUNX family transcription factor 2; sh, short hairpin; SIRT3, sirtuin 3.

Fig 3: Effect of GAS on osteogenic differentiation of LPS-stimulated hPDLSCs. (A) ALP expression in hPDLSCs was measured using ALP kit. (B) Alizarin Red staining images of hPDLSCs (magnification, x200). (C) Alizarin Red staining quantification. (D) Protein expression of ALP and Runx2 was detected by western blotting. (E) Protein expression of OCN and OPN was detected by western blotting. ***P<0.001 vs. control; #P<0.05, ##P<0.01 and ###P<0.001 vs. LPS. ALP, alkaline phosphatase; GAS, gastrodin; hPDLSCs, human periodontal ligament stem cells; LPS, lipopolysaccharide; OCN, osteocalcin; OD, optical density; OPN, osteopontin; Runx2, RUNX family transcription factor 2.

Fig 4: Osteogenesis-related biomarker expression. (A–D) Quantitative RT-PCR analysis of MSCs’ ALP, RUNX2, OCN, and OPN gene expression when cultured with GelMA, GelMA-HAP, GelMA-SN, and GelMA-HAP-SN hydrogel on days 7 and 14. (E) Immunofluorescence staining of ALP (green), RUNX2 (red), OCN (green), and OPN (red) with DAPI-labeled nuclei (blue) and phalloidin-labeled actin (opposite to the proteins); (F–I) Quantification analysis of the immunofluorescence staining results indicating ALP, RUNX2, OCN, and OPN protein expression. *P < 0.05 indicates statistical difference.

Fig 5: The expression of DDAH1 is responsive to mechanical force via TAZ/SMAD4 signaling pathway.a The typical images of immunofluorescence staining of DDAH1 (green) and DDAH2 (red) in bones of exercise-treated or tail-suspension mice. Scale bar = 100 µm. b Quantification analysis of DDAH1 and DDAH2 expression of bone parameters. ns no significance. Ground group mice, n = 5. Unload group mice, n = 5. Control group, n = 5. Exercise group, n = 5. *p < 0.05. ***p < 0.005. Data are represented as mean values ± SD. c Metabonomics of serum samples of control mice and exercise mice. d Volcano plot of different metabolites responsive to exercise treatment. e Serum samples of exercise mice or tail-suspension mice were analyzed by LC-MS assay. Ground group mice, n = 5. Unload group mice, n = 5. Control group, n = 5. Exercise group, n = 5. **p < 0.01. ***p < 0.005. Data are represented as mean values ± SD. f Western blotting analysis of the levels of phosphor-YAP, YAP, TAZ, ALP, and DDAH1 in osteoblasts treated with tension force for 6 h or not, and then cultured for 18 h, the cycle was repeated three times. g Co-IP assay of the association between SMAD4 and TAZ in simulation with tension force. h Representative immunofluorescence staining images of the association between SMAD4 (green) and TAZ (red) in simulation with tension force. Scale bars = 100 µm. i ChIP assay of SMAD4 clustering on the promoter regions of Ddah1 in stimulation with tension force. j Quantification analysis of ChIP assay. Control, n = 3. Tension, n = 3. *p < 0.05. **p < 0.01. Data are represented as mean values ± SD. k Luciferase reporter genes assay of Ddah1 from WT primary osteoblasts in stimulation with tension force by the silence of TAZ and SMAD4. All groups, n = 3. *p < 0.05. **p < 0.01. ***p < 0.005. Data are represented as mean values ± SD. The data shown in all panels were analyzed by an unpaired two-tailed Student’s t test. One-way analysis of variance (ANOVA) with post-hoc Tukey’s test was used for experiments with three or more groups.