Characterization of isolated exosomes.

<p><b>(A)</b> Scheme of exosome analysis. <b>(B)</b> Transmission electron micrograph showing exosomes isolated from the cell culture supernatant of 3 Gy-irradiated BHY cells [scale bar: 100 nm]. <b>(C)</b> Representative immunoblot of HSP70, actin, CD63 and calnexin performed with exosome lysates (EXO) and cell lysates (CP) harvested 24 hours after irradiation. DMEM medium, DMEM medium supplemented with exosome-depleted fetal calf serum as well as supernatant after ultracentrifugation were loaded as controls. <b>(D)</b> Size distribution of exosomes from non-irradiated BHY cells measured with NanoSight technology. <b>(E)</b> Relative exosome abundance of BHY exosomes isolated 24 hours after irradiation with 0, 3, 6 and 9 Gy [n = 6, p-value < 0.05].</p

Exosomes modulate the repair of DNA DSBs in irradiated recipient cells.

<p><b>(A)</b> Number of 53BP1 foci in BHY cells 1 hour after irradiation with 0 and 2 Gy and transfer of BHY exosomes isolated 24 hours after irradiation with 0 and 6 Gy [n = 5]. <b>(B)</b> Representative images of 53BP1 foci in BHY cells 6 hours after 2 Gy and transfer of BHY exosomes isolated 24 hours after irradiation with 0, 3, 6 or 9 Gy (53BP1 foci green, nuclei blue). <b>(C)</b> Number of 53BP1 foci in BHY cells 6 hours after 2 Gy and transfer of BHY exosomes isolated 24 and 48 hours after irradiation [n₁ (control; EXO 0 Gy 24 h; EXO 6 Gy 24 h) = 6, n₂ (EXO 0 Gy 48 h; EXO 3 Gy; EXO 6 Gy 48 h; EXO 9 Gy) = 3]. <b>(D)</b> Number of 53BP1 foci in FaDu cells 6 hours after 2 Gy and transfer of FaDu exosomes [n = 3]. <b>(E)</b> Number of 53BP1 foci in FaDu cells 6 hours after 2 Gy and transfer of BHY exosomes [n = 3]. <b>(F)</b> Number of 53BP1 in BHY cells after 2 Gy and transfer of destabilized BHY exosomes. Exosomes from BHY cells isolated 24 hours after irradiation with 0 and 6 Gy were treated with RNase A or a mixture of Triton and Trypsin [n₁ (control; intact) = 6; n₂ (RNase A 5 μg/μl) = 2; n₃ (RNase A 400 μg/μl; Triton + Trypsin) = 3]. For all experiments the ± SD was shown and p-values calculated on control were considered to be significant if <b>*</b> p < 0.05 and highly significant <b>**</b> if p < 0.01, while ^▲ p < 0.05 and ^▲▲ p < 0.01 indicate significant differences to EXO 0 Gy.</p

Exosomes affect proliferation, colony formation and clonogenic survival.

<p><b>(A)</b> Proliferation of cells cultivated for 3 days in medium containing exosomes isolated from irradiated or non-irradiated cells. As a control an equal amount of PBS without exosomes was added to the recipient cells. <b>(B)</b> Plating efficiency of cells cultivated for 5 days in medium containing exosomes isolated from irradiated or non-irradiated cells. As a control an equal amount of PBS without exosomes was added to the recipient cells. <b>(C)</b> Clonogenic survival of BHY cells co-cultivated with exosomes isolated from irradiated or non-irradiated cells and control cells (BHY + PBS) were incubated for 5 days after irradiation with the indicated doses [n = 3, ± SD, p-value: <b>*</b> if p < 0.05, <b>**</b> if p < 0.01 and <b>****</b> if p < 0.0001].</p

Uptake of exosomes by recipient cells.

<p>PKH67-labeled exosomes isolated from irradiated and non-irradiated BHY cells were co-cultivated with BHY cells. <b>(A)</b> Representative fluorescence microscopy images for exosome uptake after 3, 6 and 24 hours incubation. Exosomes were stained in green and nuclei were stained blue with Hoechst 33342. <b>(B)</b> Uptake of exosomes isolated from 6 Gy-irradiated (EXO 6 Gy) and non-irradiated BHY cells (EXO 0 Gy) after 3, 6, 8, 10 and 24 hours incubation. Mean fluorescence of untreated cells and cells after incubation with stained exosomes or an exosome-negative control (-EXO) is shown (n = 3). <b>(C)</b> Dependency of exosomal uptake was determined after 24 hours by using a serial dilution of an exosome preparation. <b>(D)</b> Uptake of labeled exosomes by 0, 2 and 4 Gy-irradiated recipient cells after 24 hours. In all experiments a minimum of 10,000 cells were analyzed for each sample [n ≥ 3, ± SD, p-value < 0.05].</p

Expression of miR-525-3p is up-regulated after ionizing radiation and modulation of the miR-525-3p expression effects cell survival.

<p>(A) <i>left</i> miR-525-3p expression was examined 0, 2, 4, 8, 12, 24 and 48 h after 2.5 Gy IR in the endothelial cell line EA.hy926 by quantitative real time PCR. <i>right</i> modulation of miR-525-3p results in a change in endothelial cell proliferation after IR. Endothelial cells were transfected with pre-miR-525-3p, miR-525-3p-inhibitor or scrambled control RNA, reseeded and the cell proliferation assay was performed 5d after IR. (B) HeLa cells, (C) RPE cells, (D) U2-OS cells. The mean ± s.e.m. of three independent experiments is shown. * mark significant differences between samples harvested at the 0 h timepoint compared with the indicated time point (* p<0.05, ** p< 0.01).</p

Proposed function of the most evidently changed miRNAs after UVA and UVB irradiation.

<p>Proposed function of the most evidently changed miRNAs after UVA and UVB irradiation.</p

Identification of direct targets of miR-23b using luciferase assays.

<p>(A) Relative luciferase activities after co-transfection of luciferase constructs and control miRNA or pre-miR-23b in HaCaT cells. The firefly luciferase values were normalized for transfection with renilla luciferase activity. Relative luciferase activities represent the ratio between normalized luciferase activities of pre-miR-23b and control miRNA transfected cells. The mean ± s.e.m. of three independent experiments is shown. (B) Complementarity of miR-23b sequence to the RRAS2 gene sequence. Vertical lines indicate identity between miRNA sequence and corresponding gene sequence. (C) Transcriptional change of the putative targets (RRAS2, TGFBR2 and VHL) of miR-23b in human primary keratinocytes after UVA treatment (600 kJ/m², 6h post irradiation) was analyzed via qPCR. Geometric mean of the expression of the house keeping genes: ACTB (beta actin), HPRT1 (hypoxanthine phosphoribosyltransferase 1) and TBP (TATA box binding protein) was used for normalization. Fold-change of the transcription upon UVA was obtained by setting the control as one-fold. Two-fold threshold was applied as criterion of altered transcriptional response. Error bars indicate standard deviations. N ≥ 3.</p

miRNA expression changes in primary human keratinocytes 6h after UVA irradiation (^†also changed after UVB).

<p>: miR-886-5p is a fragment of vault RNA (VTRNA2-1) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083392#pone.0083392-Stadler1" target="_blank">[60]</a>.</p

Ingenuity pathway analysis of proteins deregulated 12 h after irradiation in the absence of miR-525-3p.

<p>(A) IPA of direct and indirect miR-525-3p target proteins. The most significant network ”Cell Death and Survival, Free Radical Scavenging, Cancer” (score 26) is shown. (B) IPA of direct miR-525-3p target proteins. The most significant network “Cell Death and Survival, Organismal Injury and Abnormalities, Respiratory Disease” (score 14) is shown. Molecules in grey represent miR-525-3p target proteins. direct interaction, ------ indirect interaction.</p

Comparison of UVA and UVB deregulated miRNAs 6h after irradiation.

<p>(A) miRNAs up-regulated (red) or down-regulated (green) after UVA <u>and</u> UVB are colored. (B) Venn diagram shows the overlap between UVA- and UVB-regulated miRNAs. (C) Summary of miRNA analysis of human primary keratinocytes from two female donors after UVA and UVB irradiation. Deregulated miRNAs display a p-value < 0.05 (one sample t-test).</p