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. 2020 Oct 15;31(22):2463–2474. doi: 10.1091/mbc.E19-03-0133

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© 2020 Davies et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 3: — Apical and basolateral small EV fractions exhibit differences in number and contents. (A) NTA total counts of small EV fraction particles isolated from the apical and basolateral conditioned media revealing that a greater number of particles within the apical chamber of polarized NHCs was apparent (n = 24, p value <0.05). (B) Total RNA concentration was assessed following isolation of RNA from equal numbers of apical and basolateral small EVs. Apical EV RNA content was elevated relative to basolateral EV RNA content (n = 10, p value <0.0001). (C–E) Cholesterol level was assessed and normalized to EV number (C), protein level (D), or phospholipid level (E). Apical EVs exhibited increased cholesterol per EV (n = 10, p value <0.02) and increased cholesterol:protein (n = 6, p value <0.03) and cholesterol:phospholipid (n = 5, p value <0.04) ratios. (F) Protein concentration was assessed and normalized to EV number (n = 27). (G) Extracts generated from equal numbers of apical and basolateral EVs were resolved by SDS–PAGE and silver stained. A number of species enriched in the apical (red) or basolateral (blue) EV samples are indicated for the purpose of illustration.