Fig 2: Usp8Osx mice display impaired ossification during skeletogenesis. (A) Alizarin red/alcian blue staining of skeletal preparations of whole body, forelimbs, hindlimbs, skull, and clavicle of E18.5 Usp8fl/fl and Usp8Osx embryos (n = 3/group). Scale bar, 1 mm. (B,C) Alizarin red/alcian blue staining of skeletal preparations of hindlimbs of P0 neonates (B, n = 3/group) and E.15.5 embryos (C, n = 3/group). Scale bar, 1 mm. (D) Safranin O–stained longitudinal sections of P0 Usp8fl/fl and Usp8Osx femurs (n = 3/group). Scale bar, 250 μm (left) and 100 μm (right, enlarged insert image). (E) Immunohistochemistry (IHC) for Bglap in the trabecular bone of P0 Usp8fl/fl and Usp8Osx femurs (n = 3/group). Upper: IgG control, middle and down: IHC for Bglap. Scale bar, 100 μm.

Fig 3: Overexpression of WTAP promoted bone formation in OVX mice. (A) Schematic diagram illustrating the experimental design. (B) Verification of the efficiency of WTAP overexpression at the mRNA level on day 7 after lentivirus injection in mice. (C) Live animal fluorescence imaging analysis of luciferase expression in the WTAP-overexpressing group and empty vector-carrying group. (D) qRT-PCR analysis of ALP, COL1, and OCN mRNA levels in bone specimens from mice at 7 weeks post-injection. (E) Representative images showing the 3-dimensional trabecular architecture in distal femurs determined by micro-CT reconstruction. (F) Micro-CT measurements of BS/BV, Tb. Sp, BMD, BV/TV, Tb. N, and Tb. Th in the distal femurs of mice after treatment with WTAP-overexpressing lentivirus or empty vector-carrying lentivirus. (G) H&E staining indicated trabecular density. (H) Masson trichrome staining indicated trabecular density and collagen. Data are expressed as the mean ± SEM, *P < .05, **P < .01, ***P < .005.

Fig 4: OGN, Runx2 and OCN positive expression rates were down-regulated in the DOP models. (A) Representative immunohistochemistry (IHC) image of OGN, Runx2 and OCN staining of femur with DOP and control groups (× 100, scale bar: 200 μm, × 200, scale bar: 100 μm). (B) OGN, Runx2 and OCN positive expression rates of femur in each group. *P < 0.01, compared with control groups.

Fig 5: miR‐615‐3p directly targets FBLN1's 3′UTR, negatively regulating osteogenic differentiation of bone regeneration ability of WJCMSCs. (A) miR‐615‐3p knockdown increased ALP activity in WJCMSCs. (B, C) Alizarin red staining and quantification analysis. (D) qPCR results showed that knockdown of miR‐615‐3p upregulated the expression of OSX in WJCMSCs. (E–G) qPCR results showed that knockdown of miR‐615‐3p increased DSPP (E), DMP1 (F) and OCN (G) in WJCMSCs after mineralization induction. (H) The protein expression levels of DSPP, DMP1 and OCN on day 14 after mineralization induction. (I, J) Representative micro‐CT and the 3D reconstructed micro‐CT images of the critical‐sized defect in the WJCMSCs/Consh group and the WJCMSCs/miR‐615‐3p‐inhibitor group. (K) The relative tissue volume levels were quantified by 3D volumetric analysis. (L) Histological sections images of the critical‐sized defect, as shown by an H&E staining assay, the WJCMSCs/ miR‐615‐3p‐inhibitor group had significantly more new bone formation than the WJCMSCs/Consh group (Bar: 1 mm or 500 μm). (M) Masson staining assay showed that the amount of collagenous fibre and the area of new bone formation in the WJCMSCs/miR‐615‐3p‐inhibitor group was greater than that in the WJCMSCs/Consh group (Bar: 1 mm or 500 μm). (N–P) Immunofluorescence staining showed the DSPP, DMP1 and OCN expression (Bar: 500 μm). GAPDH served as the internal control. Student's t‐test was performed to determine statistical significance. All error bars represent the SD (n = 3 or 6). **p ≤ 0.01.