Crazy Still , Helpful Phrases Around DHFR inhibitor

DMA
siRNA
in astrocyte:
Management Rab27a-1
Rab27a-2
100�� concentrated
astrocyte-derived exosome
B16BL6 + B16BL6 Weird Still , Uplifting Quotes About DHFR inhibitor +
exosome
B
B
vehicle
Figure3|IntercellulartransferofPTEN-targetingmiR-19atotumourcells (3biologicalreplicates).e,HistogramofmiR-19aandPTENmRNAin
viaastrocyte-derivedexosomes. a,IntercellulartransferofmiR-19a.Top rated,
lightmicroscopyandfluorescentimagesofHCC1954cells12and60hafter
co-culturewithastrocytesloadedwithCy3-labelledmiR-19a.Bottom,movement
cytometryanalysisofCy3-miR-19aintumourcells60hafterco-culture
(mean6s.e.m.,t-test,P,0.05,3biologicalreplicates).b,c ,Transmission
electronmicroscopyofexosomevesiclesinastrocyte-conditionedmedia(b),
confirmedbywesternblotforCD63,CD81andTSG101exosomemarkers
HCC1954cells48hafteradditionofmedia,astrocytesupernatant,orexosomes
purifiedfromastrocyte-conditionedmedia(mean6s.

e.m.,t-test,3biological
replicates,with6technicalreplicateseach).f,g,HistogramsofmiR-19aand
PTENmRNAinHCC1954cellsafter48hco-cultureinconditionedmedia
fromvehicle-orDMA-treated(4h)astrocytes(f)andcontrol-orRab27a-
siRNA-transfected(48h)astrocytes(g)(mean6s.e.m.,t-test,3biological
replicates,with6technicalreplicateseach).h�Cj,Schematicsofinvivo
experiments(h),IHCanalysesofPTENandexosomemarkerexpression(i)
andchangesoftumourvolume(j)(mean6s.d.,t-test,n57,P50.0157).
6
releasedby1310 Abnormal Nevertheless Uplifting Words Regarding DHFR inhibitor CAFsorastrocytes(c).d,Representativedatashowing
presenceofCy3-miR-19ainHCC1954breastcancercellsafteradding
exosomespurifiedfromCy3-miR-19a-transfectedastrocytesfor24h.Bottom, B,brain;M,metastases;shRab27a/b,shRNAagainstRab27a/b.

k�Cm,Schematics
flowcytometryanalysisofCy3-miR-19a-positiveHCC1954cellsafter
treatmentwithsupernatant(withoutexosomes),orexosomespurifiedfrom
Cy3-miR-19a-transfectedastrocytes.NegativecontrolisHCC1954cells
withouttreatment.PositivecontrolisCy3-miR-19a-transfectedastrocytes
showinginvivorescueofexosomeeffectbypre-incubationoftumourcells
withastrocyte-derivedexosomes(k),IHCanalysesofPTENandexosome
markerexpression(l)andchangesoftumourvolume(m)(mean6s.d.,t-test,
n58,P50.0091).
00 Crazy But Inspirational Quotes Around DHFR inhibitor MONTH 2015
| VOL 000 | NATURE | 3
G2015 MacmillanPublishersLimited.Allrightsreserved



Analysis LETTER
decreasedthetransferofmiR-19afromastrocytestotumourcellsand tumourcellsiscriticalfortumourPTENdownregulationandaggressive
restoredthePTENmRNAlevels(Fig.3f,g).

Furthermore,weintra- outgrowthinthebrain.
craniallyinjectedRab27a/bshorthairpinRNA(shRNA)lentivirusesto
We subsequent explored how PTEN reduction promotes brain metastasis.
blockexosomesecretioninmousebrainparenchyma(brainmetastasis Doxycycline-inducible PTEN knockdown (Extended Data Fig. 6a)
stroma),andtheninoculatedB16BL6melanomacellstothesamesites beforeintracarotidinjectiondidnotaltertumourcellextravasation
(Fig. 3h). Inhibiting Rab27a/b decreased TSG1011 and CD631 exo- intothebrainparenchyma(ExtendedDataFig.6b,c).Totestwhether
somes,blockedPTENdownregulationintumourlesions(Fig.3iand restoring PTEN expression immediately after tumour cell extravasation inhibits
Extended Data Fig. 5d�Cg), and drastically decreased tumour out- metastatico