Selective Release of MicroRNA Species from Normal and Malignant Mammary Epithelial Cells
Figure 1
Differential Cellular Release and Retention of Small RNAs.
Medium conditioned by MCF7 cells for 5 days was enriched for exosomes by a filtration and ultracentrifugation protocol producing a P70 preparation. A The P70 was subjected to negative-staining EM. B The abundance of tetraspanin CD81, an exosome-marker was assessed in the filtered conditioned medium, the P70 pellet obtained by ultracentrifugation, and the supernatant (S70) using slot-blot (inset, n = 2). C The surface antigens CD81, CD63 and Mucin-1 were detected in the P70 fraction of the mammary epithelial cells using slot-blot. The absolute amount of bound antibody was quantified using standard-curves of antibody dilutions, and expressed as a percent of total antigenicity for the P70 of each cell line. The data of two replicate experiments for the indicated cell lines are shown. D Radiolabeled small RNAs isolated from MCF7 cells (c) and the extracellular preparation P70 (x) were separated by PAGE on a 12% denaturing gel. Star: Extracellular enriched RNA; Circle: Some extracellular RNAs identified by sequencing (see text and Tables S1 and S2). E Quantitation of labeled RNA species of D. The thin line indicates abundance of cellular small RNAs, whereas the thick line indicates the abundance of the extracellular miRNAs.