Fig 2: P53 is the key to beraprost-mediated effect on bone mass.A, B Volcano plots and heatmap of genes with significant changes in beraprost-stimulated M-BMSCs. C KEGG pathway analysis indicating altered function of the PI3K–AKT pathway. D GSEA showing that processes involved in negative regulation of protein modification were affected. E Western blot analysis of the transfection efficiency of p53 in M-BMSCs. F Quantitative analysis of ALP activity. n = 5; **P < 0.01, ***P < 0.001 by ANOVA with Tukey’s post-hoc test. G Quantitative RT-PCR of mRNA expression of Runx2, Alp, OCN, OSX, and Nedd4 in M-BMSCs. Results are shown as mean ± SD; n = 5; *P < 0.05, **P < 0.01, and ***P < 0.001 by ANOVA with Tukey’s post-hoc test. H Western blotting images of protein expression of pI3k, p-pI3k, AKT, p-AKT, mdm2, and p53 in M-BMSCs. I Representative images of ALP and ARS staining. J Western blot images showing protein expression of p53 in WT and p53 knockout mice. K Images of gene identification by DNA electrophoresis. L Representative images of micro-CT reconstruction of distal femurs. M Histomorphometric analysis of the distal femurs. Results are shown as mean ± SD; n = 5; *P < 0.05, **P < 0.01, and ***P < 0.001 by ANOVA with Tukey’s post-hoc test.

Fig 3: Nedd4 directly recognizes Runx2.A M-BMSCs treated with beraprost (10 µM) for 12 h were cultured with MG132 (50 mM) or NH4Cl (50 mM) for 6 h, and Runx2 protein level was analyzed by western blotting. B M-BMSCs were transfected with the indicated plasmids. Two days after transfection, the cells were treated with PBS or beraprost (10 µM) for 12 h, and then lysed using RIPA buffer. Lysates were immunoprecipitated with mouse anti-myc antibody. Western blotting was performed to analyze the levels of ubiquitination. C, D Interaction between endogenous Runx2 and Nedd4 following beraprost treatment. M-BMSCs treated with PBS or beraprost (10 µM) for 12 h were lysed with RIPA buffer. Whole-cell lysates (WCL) were used for IP with IgG, anti-Nedd4 antibody or anti-Runx2 antibody, followed by immunoblotting (IB) with the indicated antibodies. E Schematic illustration of wild-type and deletion mutants of Nedd4. F The WW domain of Nedd4 is necessary for interaction with Runx2. Myc-Runx2 was co-transfected with various flag-Nedd4 deletion mutants in M-BMSCs. WCLs were used for immunoprecipitation with anti-flag antibody, and IB with anti-flag or anti-myc antibodies. WCL was IB as a control to show the protein expression. G M-BMSCs transfected with lentiviral vector, lentiviral shRNA against Nedd4 or lentivirus encoding Nedd4 were treated with PBS or beraprost (10 µM) for 12 h. Protein expression levels of Runx2 and Nedd4 were measured by western blotting. H Knockdown of Nedd4 decreased the ubiquitination of Runx2 in vitro. M-BMSCs were transfected with the indicated plasmids. Two days after transfection, the cells were treated with PBS or beraprost (10 µM) for 12 h, and the cells were lysed with RIPA buffer. Lysates were immunoprecipitated with mouse anti-myc antibody. Western blotting was performed to analyze the levels of ubiquitination. I Schematic illustration of the role of beraprost in postmenopausal osteoporosis.

Fig 4: NEDD4 attenuates cellular damage and release of inflammatory factors in ox-LDL-induced HUVECs via regulating APEX1 expression. (A) RT-qPCR and (B) western blotting were employed to detect the interference level of APEX1 following the transfection of siRNA-APEX1-1 and siRNA-APEX1-2. (C) CCK-8 was used to test cellular activity damage. (D) An LDH kit was used to measure cytotoxic damage under the treatment of ox-LDL, overexpression of NEDD4 and interference with APEX1. (E) Detection of TNF-a, IL-1ß and IL-6 expression was conducted by RT-qPCR. **P<0.01 and ***P<0.001. NEDD4, neuronally expressed developmentally downregulated 4; ox-LDL, oxidized low density lipoprotein; HUVECs, human umbilical vein endothelial cells; APEX1, apurinic/apyrimidinic endodeoxyribonuclease 1; RT-qPCR, reverse transcription-quantitative PCR; si, short interfering; LDH, lactate dehydrogenase.

Fig 5: An illustration showing that spatial training induces decrease of Ndfip1 expression through NMDA receptor mediation, and it subsequently reduces the association between Ndfip1 and Nedd4, that results in decreased Beclin 1 and PTEN ubiquitination and enhanced spatial memory formation.