Secretion of EpCAM-exosomes, CD9-exosomes, and HSP90 by 3D aggregates of multipotent neuroendocrine adenocarcinoma cells.

Abstract

<p>(A) TEM of exosomes secreted by PC-3 cells. Scale bar, 100 nm. (B, C) Cellular (B) and exosome (C) protein concentrations per million cells. PC-3 cells were pre-cultured the four different conditions and further cultured in serum-free medium for 2 days to prepare exosome and non-exosome fractions. (D) Western blotting analysis of EpCAM, CD9, and HSP90α in cellular, exosome, and non-exosome fractions. Each protein sample per 3 x 10⁵ cells was used for analysis of exosome, per 1 x 10⁵ cells was used for analysis of non-exosome fraction, and per 2 x 10⁴ cells was used for analysis of cell lysates. (E) Western blotting analysis of E-cadherin and Vimentin. Each protein sample per 2 x 10⁴ cells was loaded. β-actin and GAPDH were analyzed as loading controls. (F) Immunocytochemistry of EpCAM, vimentin, chromogranin A (CHGA), synaptophysin (SYP), and CD34 in the 2D culture conditions. Scale bar, 50 μm. Percentages of positive cells were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191109#pone.0191109.t004" target="_blank">Table 4</a>. Photomicrographs taken at a 20 x magnification is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191109#pone.0191109.s001" target="_blank">S1 Fig</a>.</p