Fig. 7.

Release of IL-1β via exosomes.

A. VSMCs were treated with or without CaP particles (12.5 μg/mL) for 16 h with or without a pre-treatment with spiroepoxide (10 μM). A two-way ANOVA followed by Sidak's multiple comparisons test was used to compare groups. Significant differences are indicated by * (P < 0.05), n = 3, i.e. VSMC isolates from 3 different individuals (indicated by 3 different symbols).

B. Exosome secretion by VSMCs. VSMCs were incubated in 2.5%FBS/M199 media with or without CaP particles (12.5 μg/mL) and with or without R406 (1 μM) for 16 h. Conditioned media were harvested and exosomes were quantified using an anti-CD63 bead-capturing assay which detects CD63/CD81 exosomes. Statistical significance was tested by one-way ANOVA followed by Sidak's multiple comparisons test. Significant differences are indicated by **(P < 0.01), ***(P < 0.001), n = 10.

C. Western analysis of exosomes isolated by differential ultracentrifugation from conditioned media of VSMCs treated with or without CaP particles for 16 h. Exo, exosomes; VSMC, whole cell lysates. The membrane was probed with IL-1β antibodies (R and D), CD63 (BD Pharminigen), with vinculin [38] and Coomassie brilliant blue staining to demonstrate protein loading.