Fig. 3

CT-Exo protected VSMCs against calcification by promoting autophagy. (a) Representative fluorescence micrograph of PKH26-labelled CT-Exo (red) internalised by VSMCs; nuclei are shown in blue. The white scale bar is 50 μm. ARS (b) and SA-β-gal (d) staining was evaluated in VSMCs incubated with β-GP and CT-Exo for 28 and 10 days, respectively. n = 5, the black scale bar is 200 μm. (c, e) The data are presented as ratio of positive staining area. (f) ALP staining was measured in VSMCs incubated with β-GP and CT-Exo for 14 days. The black scale bar is 200 μm. (g) ALP activity. (h) RUNX2 and p53 protein expression was determined by western blotting after β-GP and CT-Exo treatment for 3 days. The data are presented as densitometric ratios normalised to β-actin (i), n = 4. (j, k) Western blots (j) and quantification (k) of p62 and LC3B in the PBS, PS and CT-Exo VSMCs, n = 4. (l) VSMCs were incubated with β-GP and CT-Exo for 72 h and then analysed by electron microscopy; a representative image is shown. Autophagosomes containing organelle remnants are highlighted by red arrows (n = 4 per group). The PS group represents the control group with only β-GP treatment. Each experiment was repeated three times. The data are presented as the mean ± standard deviation with three replicates. 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