www.sciencesignaling.org/cgi/content/full/2/1/ra81/dc1 Supplementary Materials for Delivery of MicroRNA-126 by Apoptotic Bodies Induces CXCL12- Dependent Vascular Protection Alma Zernecke,* Kiril Bidzhekov, Heidi Noels, Erdenechimeg Shagdarsuren, Lin Gan, Bernd Denecke, Mihail Hristov, Thomas Köppel, Maliheh Nazari Jahantigh, Esther Lutgens, Shusheng Wang, Eric N. Olson, Andreas Schober, Christian Weber* *To whom correspondence should be addressed. E-mail: cweber@ukaachen.de (C.W.) and alma.zernecke@virchow.uni-wuerzburg.de. (A.Z.) This PDF file includes: Published 8 December 29, Sci. Signal. 2, ra81 (29) DOI: 1.1126/scisignal.261 Fig. S1. Identification of the targets of mir-126. Fig. S2. Repression of luciferase reporter activity by, CXCL12, and mutated controls. Fig. S3. Comparative analysis of the abundance of RGS3 and. Fig. S4. Increased abundance of in transfected HUVECs. Fig. S5. CXCL12-mediated phosphorylation of ERK1/2 in HUVECs. Fig. S6. Generation of apoptotic bodies by apoptotic HUVECs. Fig. S7. Apoptotic bodies do not mobilize monocytes or neutrophils to peripheral blood. Fig. S8. Sparse colocalization of Sca-1 with macrophages in atherosclerotic lesions of mice treated with apoptotic bodies. Fig. S9. Apoptotic bodies isolated from human plaques and from mouse blood contain mir-126. Fig. S1. Apoptotic bodies specifically inhibit apoptosis and induce the proliferation of HUVECs. Table S1. The expression of mirnas in SMCs.
A target gene miranda score* CXCL12 15.944 VCAM-1 15.723 RGS3 15.32 14.578 * based on http://microrna.sanger.ac.uk/targets/v5/ B H. sapiens hsa-mir-126/mmu-mir-126 M. musculus Rgs16 5 -GCCAGUGUUUUUUGUGG -UAUGA-3 : : : : : : : : : : : 3 -GCGUAAUA AUGAGUGCC -AUGCU-5 5 - AUCAGUUCUCUUUGUGGGUAUGA-3 Fig. S1. Identification of the targets of mir-126. (A) The miranda score for highly likely targets of mir-126 are given based on algorithms according to http://microrna.sanger.ac.uk/targets/v5. (B) Schematic of potential mir-126-binding sites in mrnas for human and mouse. Complementary nucleotides are indicated by vertical bars, G:U wobble nucleotide pairs are indicated by :, and nucleotides conserved between human and mouse are indicated in green.
CXCL12 antisense antisense antisense antisense CXCL12 CXCL12-1 % repression -2-3 -4-5 ** *** -6 Fig. S2. Repression of luciferase reporter activity by, CXCL12 and mutated controls. Repression of luciferase activity in HUVECs cotransfected with pre-mir-126 and pmir-report plasmids carrying mir-126-binding sites found in the 3 UTR of, CXCL12 or with antisense sequences of or CXCL12 relative to that in HUVECs transfected with the empty pmir-control plasmid. **, P <.5; ***, P <.1; n = 3.
98 64 55 36 55 36 HUVECs Jurkat 11 1 91 1 RGS3 RGS3/β-actin (% Jurkat) /β-actin (% Jurkat) β-actin Fig. S3. Comparative analysis of the abundance of RGS3 and. The abundance of RGS3 and proteins in comparison to that of β-actin were determined by Western blotting analysis of lysates from HUVECs and Jurkat T cells. Molecular weight markers are indicated at the left margin (quantification given as a percentage relative to the abundance of β-actin and to that in Jurkat cells). One experiment representative of three experiments is shown.
control pcmv- β-actin 1 218 / β-actin (%) Fig. S4. Increased abundance of in transfected HUVECs. The abundance of protein was analyzed by Western blotting of lysates from mock-transfected HUVECs (control) and from HUVECs transfected with pcmv- and was compared to that of β-actin by densitometric analysis. One experiment representative of three experiments is shown.
Fig. S5. CXCL12-mediated phosphorylation of ERK1/2 in HUVECs. Flow cytometric analysis of the abundance of perk1/2 compared to that of total ERK1/2 indicated that CXCL12-induced phosphorylation of ERK1/2 in HUVECs was amplified by transfection with sirna against but not that against SPRED1 or of a scrambled control sirna in a process mediated by CXCR4, as evident by inhibition with AMD31. * P <.5; n = 4.
Fig. S6. Generation of apoptotic bodies by apoptotic HUVECs. The induction of apoptosis in HUVECs by serum starvation (right) as compared to controls (left) is associated with the formation and release of apoptotic bodies, as detected by scanning electron microscopy. Staining of cells with immunogold-labeled annexin-v (white dots) revealed the presence of annexin-v + detachments that formed from cytoplasmic buds and protrusions in apoptotic HUVECs, but not in untreated controls (n = 5).
Fig. S7. Apoptotic bodies (AB) do not mobilize monocytes or neutrophils to peripheral blood. Flow cytometric analysis of CD45 + CD115 + monocytes or CD45 + CD115 - Gr-1 hi neutrophils in the peripheral blood in control-treated Apoe -/- mice and in Apoe -/- mice 1 day after injection with AB (n = 5).
Sca-1 Mac-2 merge+dapi Fig. S8. Sparse colocalization of Sca-1 with macrophages in atherosclerotic lesions of mice treated with apoptotic bodies. Apoe -/- mice were fed a high-fat diet for 6 weeks and injected twice-weekly with apoptotic bodies. Images show double immunofluorescence staining for Sca-1 and Mac-2 with counterstaining of nuclei by DAPI in the aortic root (n = 7 to 8).
A human plaque AB CD31 APC 1 2 1 3 1 4 1 5 pro-mir-126 U6 31.5 28.6 37.8 38.7 Ct Ct, blank B 1 5 1 2 1 3 1 4 annexin-v FITC mouse blood AB 1 5 pro-mir-126 U6 CD31 PE-Cy-7 1 2 1 3 1 4 33. 28. 35.5 33.5 Ct Ct, blank -416 1 3 1 4 annexin-v FITC 1 5 Fig. S9. Apoptotic bodies isolated from human plaques and from mouse blood contain mir-126. CD31 + annexin-v + apoptotic bodies (AB) derived from human plaques (A) or mouse blood (B) were sorted (Q2) and the expression of mir-126 was analyzed by realtime RT-PCR. Representative FACS dot plots and gels are shown; the Ct values of AB and empty samples (blanks) are given. One experiment representative of three experiments is shown.
A HUVEC annexin-v + PI - cells (% total cells) 15 1 5 control starved starved +AB * Proliferation (% control) 125 1 75 5 25 control ** AB B Jurkat annexin-v + PI - cells (% total cells) C annexin-v + PI - cells (% total cells) 15 1 5 SMC 8 6 4 2 ns control starved starved +AB ns control cyclohexamide cyclohexamide +AB Proliferation (% control) Proliferation (% control) 125 1 75 5 25 125 1 75 5 25 control control ns AB ns AB Fig. S1. Apoptotic bodies specifically inhibit apoptosis and induce the proliferation of HUVECs. Cells were treated with or without apoptotic bodies (AB). Apoptosis was induced in HUVECs (A) and Jurkat cells (B) by serum starvation and in SMCs (C) by treatment with cyclohexamide, and cells were analyzed by staining for annexin-v and propidium iodide (PI) exclusion by flow cytometry (left panels). The proliferation of HUVECs (A), Jurkat cells (B), and SMCs (C) was assessed by cell counting (right panels). *, P <.5; **, P <.1; n = 3 to 5.
Table S1. Expression of mirnas in SMCs. The Ct values of U6, let-7c, and mir-126 in SMCs in comparison to blank controls (without cdna) as a measure for evaluating the abundance of their transcripts. This analysis revealed the marked expression of U6 and let- 7c mirnas but only trace amounts of mir-126 in SMCs. mirna Sample Ct U6 SMC 18.4 U6-31.4 let-7c SMC 22.8 let-7c - 32.4 mir-126 SMC 32.1 mir-126-32.7