Fig. 4

3-MA attenuated the pro-aging/pro-calcification preventive effect of CT-Exo in vitro and in vivo. Representative images of ARS (a) and SA-β-gal (c) staining of VSMCs that had been pre-treated with the indicated concentrations of 3-MA or RAPA for 30 min and then incubated with β-GP for 28 and 10 days, respectively. n = 5, the scale bar is 200 μm. Quantitative analysis of the percentages of ARS+ (b, in red) and SA-β-gal+ (d, in green) areas. (e) Schematic illustration of the experimental design used to assess the effects of CT-Exo and 3-MA on the vascular phenotype in VD-induced mice (n = 6 per group). (f, g) Von Kossa staining showed calcified aorta from CRTL, PS, CT-Exo, 3-MA, CT-Exo + 3-MA and RAPA mice (n = 6 per group). The black scale bar is 200 μm. (h, i) p21 expression in aorta from the six groups of mice were examined by immunohistochemistry. The black scale bar is 200 μm (n = 6 per group). The CTRL group represents the negative control group with only PBS treatment. The PS group represents the positive control group with only β-GP treatment. The data are presented as the mean ± standard deviation. The data were analysed with one-way ANOVA with the Bonferroni post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001