Journal: American Journal of Translational Research

Article Title: Inorganic phosphate-osteogenic induction medium promotes osteogenic differentiation of valvular interstitial cells via the BMP-2/Smad1/5/9 and RhoA/ROCK-1 signaling pathways

doi:

Figure Lengend Snippet: Phosphorylation of Smad1/5/9 during VICs osteogenic differentiation. A. Cells were stimulated with IP-OIM for 15, 30, 60, and 120 min to detect Smad1/5/9 phosphorylation levels. B. The inhibitory efficiency of the Smad1/5/9-specific inhibitor Compound C (CC) was determined by pre-treating VICs with varying concentrations of Compound C. C. ALP activity was measured on the 6th day of co-culture with Compound C (N=3; *P<0.05, **P<0.001). D. ALP staining was performed on the 6th day. E. Detection of calcium content on the 9th day of Compound C treatment (N=3; *P<0.05, **P<0.001). F. Alizarin Red staining of cells on the 9th day. G. Phosphorylation levels of Smad1/5/9 in siRNA-transfected VICs stimulated with IP-OIM for 60 min.

Article Snippet: Subsequently, three working doses of the Smad1/5/9-specific inhibitor Compound C (MCE, New Jersey, USA) were added into the complete medium, and the VICs were pre-treated with these for 1 day.

Techniques: Phospho-proteomics, Activity Assay, Co-Culture Assay, Staining, Transfection

Journal: Scientific reports

Article Title: EIF4A3 enhances the viability, invasion and osteogenic differentiation of BMSCs via the USP53/SMAD5 pathway.

doi: 10.1038/s41598-025-86048-1

Figure Lengend Snippet: Fig. 1. Effect of SMAD5 knockdown on osteogenic differentiation of BMSCs. (A) WB analysis was used to detect SMAD5 protein levels in BMSCs cultured with GM and OM at days 0, 6 and 12. (B–I) BMSCs were transfected with sh-NC/sh-SMAD5 and then induced with OM. (B) SMAD5 protein level was tested by WB analysis. (C) Cell viability was assayed using CCK8. (D, E) Cell invasion was assessed by transwell assay. (F) ALP activity was measured by ALP Assay Kit. (G, H) ARS staining was performed to examine mineralization levels. (I) The protein expression levels of OCN, Runx2, OPN and BSP were detected by WB analysis. *P < 0.05.

Article Snippet: Besides, BMSCs transfected with sh-NC/shUSP53 and then treated with proteasome inhibitor MG132 (10 μmol/L; HY-13259, MedChemExpress) for 24 h to explore whether USP53 regulated SMAD5 expression through deubiquitinating.

Techniques: Knockdown, Cell Culture, Transfection, Transwell Assay, Activity Assay, ALP Assay, Staining, Expressing

Journal: Scientific reports

Article Title: EIF4A3 enhances the viability, invasion and osteogenic differentiation of BMSCs via the USP53/SMAD5 pathway.

doi: 10.1038/s41598-025-86048-1

Figure Lengend Snippet: Fig. 2. Effect of SMAD5 overexpression on osteogenic differentiation of BMSCs. BMSCs were transfected with pcDNA/SMAD5 overexpression vectors and then induced with OM. (A) SMAD5 protein level was tested by WB analysis. Cell viability, invasion, ALP activity, mineralization levels, and the protein levels of OCN, Runx2, OPN and BSP were measured using CCK8 (B), transwell assay (C), ALP Assay Kit (D), ARS staining (E) and WB analysis (F). *P < 0.05.

Article Snippet: Besides, BMSCs transfected with sh-NC/shUSP53 and then treated with proteasome inhibitor MG132 (10 μmol/L; HY-13259, MedChemExpress) for 24 h to explore whether USP53 regulated SMAD5 expression through deubiquitinating.

Techniques: Over Expression, Transfection, Activity Assay, Transwell Assay, ALP Assay, Staining

Journal: Scientific reports

Article Title: EIF4A3 enhances the viability, invasion and osteogenic differentiation of BMSCs via the USP53/SMAD5 pathway.

doi: 10.1038/s41598-025-86048-1

Figure Lengend Snippet: Fig. 3. Effect of USP53 on SMAD5 expression. (A) The ubibrowser website revealed the regulation of USP53 on SMAD5. (B–D) BMSCs were transfected with sh-NC/sh-USP53. (B) USP53 protein level was tested by WB analysis. (D) qRT-PCR and WB analysis were used to detect SMAD5 mRNA and protein levels. (E) Co-IP was performed to measure the interaction between USP53 and SMAD5. (F) SMAD5 protein level was detected by WB analysis in BMSCs transfected with pcDNA/USP53 overexpression vector and treated with CHX at 0, 3, 6, 9, and 12 h. (G) SMAD5 protein level was tested using WB analysis in BMSCs transfected with sh-NC/ sh-USP53 and treated with MG132. (H) Ubiquitination analysis was performed in BMSCs transfected with or without sh-NC/sh-USP53. *P < 0.05.

Article Snippet: Besides, BMSCs transfected with sh-NC/shUSP53 and then treated with proteasome inhibitor MG132 (10 μmol/L; HY-13259, MedChemExpress) for 24 h to explore whether USP53 regulated SMAD5 expression through deubiquitinating.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Over Expression, Plasmid Preparation, Ubiquitin Proteomics

Journal: Scientific reports

Article Title: EIF4A3 enhances the viability, invasion and osteogenic differentiation of BMSCs via the USP53/SMAD5 pathway.

doi: 10.1038/s41598-025-86048-1

Figure Lengend Snippet: Fig. 4. Effect of USP53 and SMAD5 on osteogenic differentiation of BMSCs. BMSCs were co-transfected with sh-NC/sh-USP53 and pcDNA/SMAD5 overexpression vector in OM. (A) WB analysis was used to test SMAD5 protein level. CCK8 (B), transwell assay (C), ALP Assay Kit (D), ARS staining (E, F) and WB analysis (G) were performed to measure cell viability, invasion, ALP activity, mineralization levels, and the protein levels of OCN, Runx2, OPN and BSP. *P < 0.05.

Article Snippet: Besides, BMSCs transfected with sh-NC/shUSP53 and then treated with proteasome inhibitor MG132 (10 μmol/L; HY-13259, MedChemExpress) for 24 h to explore whether USP53 regulated SMAD5 expression through deubiquitinating.

Techniques: Transfection, Over Expression, Plasmid Preparation, Transwell Assay, ALP Assay, Staining, Activity Assay

Journal: Scientific reports

Article Title: EIF4A3 enhances the viability, invasion and osteogenic differentiation of BMSCs via the USP53/SMAD5 pathway.

doi: 10.1038/s41598-025-86048-1

Figure Lengend Snippet: Fig. 5. Effect of EIF4A3 on USP53 expression. (A) The ENCORI websites analyzed the interaction of USP53 and EIF4A3. (B) The interaction between USP53 and EIF4A3 was detected by Co-IP. (C) BMSCs were transfected with sh-NC/sh-EIF4A3/pcDNA/EIF4A3 overexpression vector, and WB analysis was used to determine EIF4A3 protein level. (D, E) Act D assay was performed to measure the stability of USP53 mRNA. (F) WB analysis for the detection of USP53 protein expression. *P < 0.05.

Article Snippet: Besides, BMSCs transfected with sh-NC/shUSP53 and then treated with proteasome inhibitor MG132 (10 μmol/L; HY-13259, MedChemExpress) for 24 h to explore whether USP53 regulated SMAD5 expression through deubiquitinating.

Techniques: Expressing, Co-Immunoprecipitation Assay, Transfection, Over Expression, Plasmid Preparation

Journal: Scientific reports

Article Title: EIF4A3 enhances the viability, invasion and osteogenic differentiation of BMSCs via the USP53/SMAD5 pathway.

doi: 10.1038/s41598-025-86048-1

Figure Lengend Snippet: Fig. 6. Effect of EIF4A3 and USP53 on osteogenic differentiation of BMSCs. BMSCs were co-transfected with sh-NC/sh-EIF4A3 and pcDNA/USP53 overexpression vector in OM. (A) USP53 protein level was detected by WB analysis. Cell viability and invasion were measured by CCK8 (B) and transwell assay (C). (D) ALP Assay Kit was used to tested ALP activity. (E–G) Mineralization levels and the protein levels of OCN, Runx2, OPN and BSP were measured using ARS staining and WB analysis. *P < 0.05.

Article Snippet: Besides, BMSCs transfected with sh-NC/shUSP53 and then treated with proteasome inhibitor MG132 (10 μmol/L; HY-13259, MedChemExpress) for 24 h to explore whether USP53 regulated SMAD5 expression through deubiquitinating.

Techniques: Transfection, Over Expression, Plasmid Preparation, Transwell Assay, ALP Assay, Activity Assay, Staining

Journal: Scientific reports

Article Title: EIF4A3 enhances the viability, invasion and osteogenic differentiation of BMSCs via the USP53/SMAD5 pathway.

doi: 10.1038/s41598-025-86048-1

Figure Lengend Snippet: Fig. 7. EIF4A3 affected SMAD5 expression via mediating USP53. (A) WB analysis was used to measure SMAD5 protein level in BMSCs co-transfected with sh-NC/sh-EIF4A3 and pcDNA/USP53 overexpression vector. (B) Summary diagram of this study. *P < 0.05.

Article Snippet: Besides, BMSCs transfected with sh-NC/shUSP53 and then treated with proteasome inhibitor MG132 (10 μmol/L; HY-13259, MedChemExpress) for 24 h to explore whether USP53 regulated SMAD5 expression through deubiquitinating.

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation