Fig. 9.

Nano-HAp promoted release of exosomes which contained LC3 and LAMP1. A) Total protein was assessed in the Exos that were purified from the conditioned medium of VSMCs treated with or without S-HAp. B) Western Blot analysis for detection of CD9, LC3 and LAMP1 expression in the purified exosomes. Relative protein levels were quantified in comparison with the control. C) Calcium content in Exos was measured. D) Total protein was assessed in the Exos that were purified from the conditioned medium of VSMCs that were treated with 100 μg/mL S-HAp or/and 10 μm/L GW4869 for 3 days. E) Western blot analysis of CD9, LC3B and LAMP1. F) Analysis of calcium content of Exos that were treated with S-HAp or/and GW4869 for 5 days. Data are expressed as the mean ± SD. (*vs control, #vs HAp, ∗p < 0.05, ∗∗ p < 0.01, ***p < 0.001, #p < 0.05). G) Alizarin red staining for calcification detection in VSMCs that were with S-HAp or/and GW4869 for 14 days. The bar graphs show the quantification of Alizarin red and the calcium content in the cells. Data are expressed as the mean ± SD. (***p < 0.001). H) FACS analysis of Exos from the conditioned medium of VSMCs treated with or without 100 μg/mL fluorexon-labeled S-HAp. The fluorexon labeled S-HAp was appointed as a positive control.