Fig 2: Validation of UBE2E3 expression.(A) The expression of UBE2E3 in 7862 normal tissues of GTEx. (B) The correlation analysis of UBE2E3 with osteogenesis related genes in GTEx. (C) The correlation analysis of COL1A1 and UBE2E3 in GTEx. (D, E) Heatmap of single cell sequencing of rat BMSCs. (F) UBE2E3 expression of human BMSCs from GSE35956 and GSE35958. (G) qRT-PCR analyses of UBE2E3 levels in old mouse BMSCs and young mouse BMSCs. (H) Western blot analyses of UBE2E3 levels in old mouse BMSCs and young mouse BMSCs. (I) qRT-PCR analyses of UBE2E3 levels in increasing numbers of passages of mice BMSCs. (J) Western blot analyses of UBE2E3 levels in increasing numbers of passages of mice BMSCs. Error bars showed standard deviation. *P < 0.05, ***P < 0.001.

Fig 3: Overexpression of UBE2E3 attenuated cell senescence as well as enhancing osteogenic differentiation of old BMSCs.(A) Overexpression efficiency of UBE2E3 was verified by qRT-PCR. (B) Overexpression efficiency of UBE2E3 was verified by western blot. (C) ß-Gal staining and quantification of senescent BMSCs in UBE2E3 overexpression group and the control group. (D) The relative mRNA expression of p16 and p21 between UBE2E3 overexpression group and the control group. (E) ALP and ARS staining results in UBE2E3 overexpression group and the control group, with the quantitative analysis. (F) The relative mRNA expression of RUN2, ALP, SP7 and BGLAP between UBE2E3 overexpression group and the control group. Error bars showed standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Knockdown of UBE2E3 accelerated cell senescence and inhibited osteogenic differentiation of young BMSCs.(A) Knockdown efficiency of UBE2E3 was verified by qRT-PCR. (B) Knockdown efficiency of UBE2E3 was verified by western blot. (C) ß-Gal staining and quantification of senescent BMSCs in siUBE2E3 group and siNC group. (D) The relative mRNA expression of p16 and p21 between siUBE2E3 group and siNC group. (E) ALP and ARS staining results in siUBE2E3 group and siNC group, with the quantitative analysis. (F) The relative mRNA expression of RUN2, ALP, SP7 and BGLAP between siUBE2E3 group and siNC group. Error bars showed standard deviation. *P < 0.05, **P < 0.01.

Fig 5: The schematic of UBE2E3 regulating senescence and osteogenic differentiation of BMSCs during aging.High expression of UBE2E3 contributes to nuclear translation of Nrf2 and its high activity. With age, the expression of UBE2E3 decreases and nuclear accumulation of Nrf2 and its activity are restrained, leading to cell senescence and reduced osteogenic differentiation potential of BMSCs.