Fig. 5

Rab27A knockdown in breast cancer cells inhibits DHA-induced exosome transfer. The expression of Rab27A was knocked down in MCF7 CD63-GFP and MDA-MB-231 CD63-GFP breast cancer cell lines by transduction with a lentivirus expressing dsRed2 and two different shRNAs targeting Rab27A. a Bright field (left) and fluorescent microscopic images (right) of CD63-GFP (green), shRab27A-dsRed2 (red) and Hoescht nuclear stain (blue) (40× magnification). Rab27A protein and mRNA knockdown was confirmed by western blot (b) and qRT-PCR (c). The fold change in expression levels of Rab27A relative to an endogenous control was calculated using the ΔΔCt method. Data in bar graphs represent mean ± SEM *, p < 0.001 using Student’s t test. d The quantity of exosomes secreted from breast cancer cells was measured by BCA assay and represented as the μg/μl of whole exosome per 10⁶ cells, relative to wildtype. Data in bar graphs represent mean ± SEM (n = 2). e MCF7 and MDA-MB-231 cells expressing CD63 or shRab27A-CD63 were grown in a transwell insert (0.4 μm) and co-cultured with EA.hy926 cells plated on ECMatrix for 24 h. Following treatment of the breast cancer cells in the upper chamber with 100 μM DHA for 24 h the number of branch points formed by the EA.hy926 cells were counted. Data in bar graphs represent mean ± SEM (n = 3-6) *, p < 0.05; **, p < 0.01 using Student’s t test. f MCF7 cells expressing CD63 or shRab27A-CD63 were grown in a transwell insert (0.4 μm) and co-cultured with EA.hy926 cells plated in a 24-well plate. Following treatment of the breast cancer cells in the upper chamber with 100 μM DHA for 24 h total RNA was extracted from the EA.hy926 cells, reverse transcribed and expression of miR-23b and miR-320b was measured by qRT-PCR. The fold change in expression levels was calculated using the ΔΔCt method. Fold-change values are normalized to control sample and expressed relative to no breast cancer cells (−BC). Data in bar graphs represent mean ± SEM (n = 3) *, p ≤ 0.05; **, p < 0.001 using Student’s t test