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1 Supplementary Figure 1. Characterization of a functional Ago2-miRNA complex in EVs. (a) EVs derived from iRBCs contain no RNA species other than small RNAs. RNA samples were prepared from EVs and analyzed by Agilent Bioanalyzer RNA Chip. Only small RNA species < 200 nucleotides are detected. FU=Fluorescence units. Characterization of a functional Ago2-

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miRNA complex in EVs: 3’UTR degradation activity (b,c). HEK293 cells transiently expressing a Renilla luciferase reporter gene with binding site

complementary to miR-451a, let-7b, miR-15a in the 3’ untranslated region of the reporter cassette were incubated for 48 h prior to luciferase activity

measurements, either without (b) or with EVs derived from iRBCs (c). miR-451a reporter is only significantly reduced upon addition of EVs, while let-7b, miR-15a and miR-106a are also in the control suggesting endogenous activity of these miRNAs in HEK293 cells, as described (Hafner et al). Results are normalized based on constitutive Firefly luciferase activity, and expressed as mean (± SEM) percentage of control (n = 3 experiments). *p < 0.05; **p < 0.01; ***p < 0.005 versus baseline or control (Student’s t test). (d) Ago2 association of miRNAs.

Protein extracts derived from purified EVs were subjected to immuno-

precipitation (IP) using anti-Ago2 antibody, followed by Let7-b, miR-15a, miR- 106b, miR-222 quantification using qRT-PCR. Results are normalized by the 2^- Ct method, using RNU6 as a reference and expressed as percentage of the input (mean +/- SEM; n=4 experiments).

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3 Supplementary Figure 2. Predicted miR-451a target sites in CAV-1 and ATF2. Schematic representation of the 3’UTR of ATF2 (a) and CAV-1 (b).

Predicted interactions of miR-451a with their binding sites are shown, with sequences of mature miR-451a aligned to these target sites highlighted.

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Supplementary Figure 3. Transcriptional dependence of ATF2 and CAV-1 expression levels in hBMECs. qRT- PCR analysis of CAV-1 (left) and ATF2 (right) levels in BMECs pre-incubated for 7 hours with α-amanitin, followed by incubation for 24 h with EVs (100 µg of total protein/ml). Relative changes normalized to 18 S rRNA presented as fold changes. Data represent the mean± SD (n = 4).

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5 Supplementary Figure 4. Full, uncropped images of immunoblots shown in the main figure 2a and d. Molecular weight markers (BenchMark Pre-stained Protein Ladder, Thermofisher) are indicated on the left of the blots.

A B C D

115- 82- 64-

49- 37- 26-19- 15-

115- 82- 64-

49- 37- 180-

115- 82- 64- 180-

49- 37- 26-19-

64-

49- 37- 26-19- 15- E

115- 82.2- 64.2- 48.8- 37.1- 25.9- 19.4- 14.8- 115- 82.2- 64.2- 48.8- 37.1- 25.9- 19.4- 14.8-

115- 82.2- 64.2- 48.8- 37.1- 25.9- 19.4- 14.8- F

G

H

Supplementary Figure 4

SI Figure 2a

SI Figure 2d

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Supplementary Figure 5. Full, uncropped images of immunoblots shown in the main figure 3a and e. Molecular weight markers (BenchMark Pre-stained Protein Ladder, Thermofisher for A and B and Prestained Protein Ladder V, Geneaid for C and D) are indicated on the left of the blots.

115- 82.2- 64.2- 48.8- 37.1- 25.9- 19.4- 14.8-

115- 82.2- 64.2- 48.8- 37.1- 25.9- 19.4- 14.8-

Supplementary Figure 5

A B

C D

22- 35- 75-63-

48- 100-

22- 35- 75-63-

48- kDa

kDa kDa

kDa SI Figure 3a

SI Figure 3e

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7 Supplementary Figure 6. Full, uncropped images of immunoblots shown in the main figure 8b and d. Molecular weight markers (Prestained Protein Ladder V, Geneaid) are indicated on the left of the blots.

22- 70- 53-

41- 30-

22- 70- 53-

41- 30-

22- 70- 53-

41- 30-

A B C

10- 130- 70- 55- 35- 25-15-

10- 130- 70- 55- 35- 25-15-

D E

Supplementary Figure 6

kDa kDa kDa

kDa kDa

SI Figure 8b

SI Figure 8d

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Supplemental Table 1. Predicted miR-451a target genes. Shown are targets based on 4 different prediction software packages: Targetscan, microcosm, microrna.org and DIANAlab.

Gene RefSeq ID Targetscan Microscom microrna.org

DIANA Lab

ADAM28 NM_014265.4 1

ATF2 NM_001880 1 1 1

CAV-1 NM_001172895.1 1 1

EREG NM_001432.2 1

FBLN5 NM_006329.3 1 1 1

GRSF1 NM_001098477.1 2 2 3

KLF11 NM_001177716.1 1 1

MST4 NM_001042452.1 1

OSR NM_145260.2 1 1 1 1

PHACTR1 NM_001242648.1 2

PSMB8 NM_004159.4 1 1 1

PTTG1IP NM_001286822.1 1 2

RAB14 NM_016322.3 2 2

S1PR2 NM_004230.3 1 1

SAMD4B NM_018028.2 1 1 1

SNRNP70 NM_001009820.1 1

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