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. Author manuscript; available in PMC: 2016 May 5.
Published in final edited form as: Nature. 2015 Oct 19;527(7576):100–104. doi: 10.1038/nature15376

Extended Data Figure 5. Inhibition of exosome release by DMA, Rab27a siRNA or Rab27 shRNAs.

Extended Data Figure 5

a, Exosome-releasing inhibitor (DMA) treatment reduced exosome secretion from astrocytes compared to vehicle treated astrocytes. Astrocytes were treated with DMA (25μg/ml) or vehicle for 4 hours; exosomes were concentrated from astrocyte-conditioned media and total proteins from exosomes were examined by BCA assay (normalized to total cell numbers) (mean ± s.e.m., t-test). b, Knockdown of Rab27a in astrocytes by siRNA. Two siRNAs targeting mouse Rab27a were transiently transfected into astrocytes and mRNA level of Rab27a was examined by RT-PCR 48 hours after transfection (mean ± s.e.m., t-test). c, Knocking down Rab27a in astrocytes inhibited exosome release. 48 hours after Rab27a-targeting siRNAs were transfected, exosomes were collected from astrocyte-conditioned media and total proteins from exosomes were examined by BCA assay (normalized to total cell numbers) (mean ± s.e.m., t-test). d, Histogram showing relevant changes of Rab27a and Rab27b mRNA level in primary astrocytes infected with pLKO.shRab27a or pLKO.shRab27b virus (mean ± s.e.m., t-test, P<0.001, 3 biological replicates, with 3 technical replicates each). e, Change of exosome protein level detected in astrocyte-conditioned media from cells infected by pLKO.shRab27a or pLKO.shRab27b virus by BCA assay (normalized to total cell numbers) (mean ± s.e.m., t-test, P<0.001, 3 biological replicates, with 3 technical replicates each). f-g. IHC analysis showing the expression level of Rab27a, Rab27b (f) and exosome marker expression CD63 (g) in the brain tissue derived from mice injected with control lentivirus or Rab27a/b shRNA lentiviruses and subsequently intracranially injected with B16BL6 cells. h-i. IHC analysis showing the expression level of Rab27a and Rab27b (h) and exosome marker expression CD63 (i) in the brain tissue derived from mice injected with Rab27a/b shRNA lentiviruses and subsequently intracranially injected with B16BL6 cells with vehicle or B16BL6 cells with astrocyte derived exosomes.