Fig 2: TZAP expression increased with pMSC aging (P2, P6 and P10). The rates of cell growth and EdU incorporation were assayed by a CCK8 (a) assay and an EdU assay (b), respectively. Bar = 100 µm. c Bar = 100 µm. The osteogenic and adipogenic potential of pMSCs at different passages was assessed by alizarin red S staining and oil red O staining, respectively, after induction of differentiation. d The expression of bone markers (Alp, Runx2, and Col1a1) and adipogenic markers (Cebpa) was assessed by qRT-PCR after adipogenic and osteogenic induction, respectively, in pMSCs at different passages. e Bar = 100 µm. Comparison of the SA-ß-gal staining profiles of pMSCs at different passages. f Protein levels of TZAP, P16INK14 (P16) and P21 in pMSCs at different passages were compared through western blot analysis. g The mRNA levels of TZAP, p16INK14 (p16) and p21 in pMSCs at different passages were detected by qRT-PCR. All data are represented as the mean ± SEM. n = 3. *p < 0.05; **p < 0.01; #p < 0.001, tested by one-way ANOVA

Fig 3: TZAP negatively regulates cell proliferation through the p53 pathway. Comparison of western blot detection of different intracellular protein levels (TZAP, P21, P53, ARF, and MDM2) in pMSCs with TZAP overexpression (OE) and TZAP knockout (KO)

Fig 4: Overexpression of TZAP in early-passage MSCs decreased proliferation and differentiation potential. a The mRNA levels of TZAP and senescence-associated markers (p21 and p16) in pMSCs were assessed by qRT-PCR after transduction with control plasmids (CTR) or TZAP overexpression vectors (TZAP-OE). b Protein levels of TZAP, P16INK14 (P16) and P21 in pMSCs were compared through western blot analysis after TZAP overexpression. c The effect of overexpression of TZAP on the cell growth rate. d Bar = 100 µm. The EdU incorporation rate of pMSCs with TZAP overexpression compared to that of the controls. e Bar = 100 µm. Differentiation of pMSCs with TZAP overexpression was assessed by oil red O staining after adipogenic induction or by alizarin red S staining after osteogenic induction. f The relevant expression levels of bone markers (Alp, Runx2, and Col1a1) and adipogenic markers (Cebpa) were assessed by qRT-PCR and compared between pMSCs with TZAP overexpression and the control cell line. g PPAR? expression was examined by western blotting upon TZAP overexpression in pMSCs. h Bar = 100 µm. SA-ß-gal staining of pMSCs with TZAP overexpression compared to that of the control. All data are represented as the mean ± SEM. n = 3. *p < 0.05; **p < 0.01; #p < 0.001, tested by one-way ANOVA