Fig. 3.

Exosomes derived from MT-treated ECs were responsible for the anti-calcification and anti-senescence effects of melatonin. (A–F) VSMCs were cultured with EC's vehicle-induced CM (Veh-ECs-CM), melatonin-induced CM (MT-ECs-CM), and EVs-depleted MT-CM (MT-ECs-Exo free-CM), and GW4869 pretreated MT-CM (MT-ECs-GW4869-CM). (A)The expression of Runx2, BMP2, P21 of VSMCs was determined by Western blot. (B) The ALP activity in VSMCs was measured by an ALP kit. (C) The calcium content in VSMCs was measured by the o-cresolphthalein method. (D) The calcium nodules in VSMCs was measured by Alizarin red S staning (upper panel, scale bar = 200 μm), and the senescence VSMCs was measured by SA-β-Gal staining (lower panel, scale bar = 200 μm). (E) The quantification results of mineralized nodules staining in figure D. (F) The quantification results of senescence VSMCs in figure D. (G) The expression of Runx2, BMP2, P21 of VSMCs incubated with Veh-ECs-Exos and MT-ECs-Exos was determined by Western blot. (H) The ALP activity in VSMCs incubated with Veh-ECs-Exos or MT-ECs-Exos was measured by an ALP kit. (I) The calcium content in VSMCs treated with Veh-ECs-Exos or MT-ECs-Exos was measured by the o-cresolphthalein method. (J) The calcium nodules in VSMCs treated with Veh-ECs-Exos or MT-ECs-Exos was measured by Alizarin red S staining (upper panel, scale bar = 200 μm), the senescence VSMCs was measured by SA-β-Gal staining (lower panel, scale bar = 200 μm). (K) The quantification results of mineralized nodules staining in figure J. (L) The quantification results of senescence VSMCs in figure J. Three independent experiments were performed, and representative images were shown. Data are shown as mean ± SEM. Statistical analysis was performed by one-way ANOVA with Tukey's multiple-comparison test (B-F) and unpaired two-tailed Student's t-test(H-L). ns: no significance, *p < 0.05, **p < 0.01.