Journal: International Dental Journal

Article Title: FBXO5 alleviates apical periodontitis by facilitating TP53 protein degradation

doi: 10.1016/j.identj.2025.103948

Figure Lengend Snippet: FBXO5 promotes TP53 degradation through ubiquitination. (A) UbiBrowser was used to identify downstream targets of FBXO5. Circular nodes represented proteins predicted as FBXO5 substrates by UbiBrowser. Orange lines indicated domain-pair evidence, purple lines indicated GO term-pair evidence, green lines indicated E3-recognising motif evidence, and black lines indicated network-loop evidence. TP53 was highlighted with a red circle as a predicted substrate of FBXO5. (B-C) RT-qPCR and western blotting were employed to assess the mRNA and protein expression levels of TP53 in hSCAPs transfected with shNC or shFBXO5 (effect size = 0.57 for B, 5.87 for C). (D) Co-IP was performed to analyse the interaction between FBXO5 and TP53. (E) The protein level of TP53 was analysed by Western blotting in hSCAPs transfected with shNC or shFBXO5, followed by CHX treatment at various time points (0 hours, 4 hours, 8 hours, 12 hours). (F) Ubiquitination of TP53 was evaluated using Co-IP analysis after FBXO5 knockdown. Data were presented as mean ± SD. ns, no significance. Exact P values were indicated in the figure, P < .05 was considered statistically significant (Unpaired t-test for B-C).

Article Snippet: After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with anti-FBXO5 antibody (1:1000, ab187144, Abcam), anti-TP53 antibody (1:3000, 10442-1-AP, Proteintech, Wuhan, China), anti-runt-related transcription factor 2 (RUNX2) antibody (1:2000, 20700-1-AP, Proteintech), anti-DLX5 antibody (1:1000, 10592-1-AP, Proteintech), anti-osterix (OSX) antibody (1:1000, 28694-1-AP, Proteintech), anti-osteocalcin (OCN) antibody (1:1000, ab133612, Abcam), or anti-GAPDH antibody (1:1000, ab9485, Abcam).

Techniques: Ubiquitin Proteomics, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Co-Immunoprecipitation Assay, Knockdown

Journal: International Dental Journal

Article Title: FBXO5 alleviates apical periodontitis by facilitating TP53 protein degradation

doi: 10.1016/j.identj.2025.103948

Figure Lengend Snippet: FBXO5 enhances hSCAPs proliferation by suppressing TP53 expression. (A) The hSCAPs transfected with oeNC, oeFBXO5, or oeTP53 were incubated in osteogenic induction medium for 14 days. The expression of FBXO5 (effect size = 5.77) and TP53 (effect size = 3.66) were evaluated using western blotting. (B) The hSCAPs were transfected with oeNC, oeFBXO5, or oeFBXO5+oeTP53 and subsequently cultured in osteogenic induction medium for 14 days. Cell viability was assessed through the CCK-8 assay (effect size = 2.34). (C) Proliferation of cells was determined by the EdU assay (effect size = 2.49). Scale bar, 100 µm. Data were presented as mean ± SD. ns, no significance, Exact P values were indicated in the figure, P < .05 was considered statistically significant (Ordinary one-way ANOVA with Tukey's multiple comparisons test).

Article Snippet: After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with anti-FBXO5 antibody (1:1000, ab187144, Abcam), anti-TP53 antibody (1:3000, 10442-1-AP, Proteintech, Wuhan, China), anti-runt-related transcription factor 2 (RUNX2) antibody (1:2000, 20700-1-AP, Proteintech), anti-DLX5 antibody (1:1000, 10592-1-AP, Proteintech), anti-osterix (OSX) antibody (1:1000, 28694-1-AP, Proteintech), anti-osteocalcin (OCN) antibody (1:1000, ab133612, Abcam), or anti-GAPDH antibody (1:1000, ab9485, Abcam).

Techniques: Expressing, Transfection, Incubation, Western Blot, Cell Culture, CCK-8 Assay, EdU Assay

Journal: International Dental Journal

Article Title: FBXO5 alleviates apical periodontitis by facilitating TP53 protein degradation

doi: 10.1016/j.identj.2025.103948

Figure Lengend Snippet: FBXO5 promotes osteogenic differentiation of hSCAPs by negatively regulating TP53. The hSCAPs were transfected with oeNC, oeFBXO5, or oeFBXO5+oeTP53 and then cultured in osteogenic induction medium for 14 days. (A-B) ALP staining and enzyme activity assay were conducted to assess ALP activity (effect size = 4.90 and 2.84, respectively). Scale bar, 100 µm. (C) ARS staining was performed to detect mineralisation, and the stained area (%) was quantified using ImageJ software (effect size = 3.51). Scale bar, 100 µm. (D) Western blotting was used to examine the protein expression levels of RUNX2 (effect size = 4.85), DLX5 (effect size = 3.03), OSX (effect size = 8.75), and OCN (effect size = 10.58). Data were presented as mean ± SD. Exact P values were indicated in the figure, P < .05 was considered statistically significant (Ordinary one-way ANOVA with Tukey's multiple comparisons test).

Article Snippet: After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with anti-FBXO5 antibody (1:1000, ab187144, Abcam), anti-TP53 antibody (1:3000, 10442-1-AP, Proteintech, Wuhan, China), anti-runt-related transcription factor 2 (RUNX2) antibody (1:2000, 20700-1-AP, Proteintech), anti-DLX5 antibody (1:1000, 10592-1-AP, Proteintech), anti-osterix (OSX) antibody (1:1000, 28694-1-AP, Proteintech), anti-osteocalcin (OCN) antibody (1:1000, ab133612, Abcam), or anti-GAPDH antibody (1:1000, ab9485, Abcam).

Techniques: Transfection, Cell Culture, Staining, Enzyme Activity Assay, Activity Assay, Software, Western Blot, Expressing