Supplementary Information
Leukemia cell to endothelial cell communication via exosomal miRNAs
Tomohiro Umezu1, Kazuma Ohyashiki1,2, Masahiko Kuroda3, and Junko H. Ohyashiki4
1Department of Molecular Science, Tokyo Medical University, Tokyo, Japan
2First Department of Internal Medicine, Tokyo Medical University, Tokyo, Japan
3Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan
4Institute of Medical Science, Tokyo Medical University, Tokyo, Japan
Supporting Materials and Methods
Preparation of exosomes by ultracentrifugation
K562 cells or K562/Cy3-miR-92a cells (K562 cells transfected with Cy3-labeled pre-miR-92a) were pre-cultured in serum-free AIM V medium (Invitrogen). The cells were cultured in 200 ml of AIM V medium (20100-mm culture dishes). For exosome preparation, the culture media were collected and centrifuged at 2,000 × g for 10 min at room temperature. The resultant supernatant was centrifuged again at 12,000 × g for 30 min at room temperature, then the supernatant was ultracentrifuged at 110,000 × g for 70 min at 4°C. The pellets were washed with PBS after ultracentrifugation and used for microarray analysis.
RNA isolation for miRNA microarray analysis
The exosome pellets were obtained by using Exoquick (System Biosciences) or ultracentrifugation from the culture medium of K562 cells. Total RNA in the exosome was isolated using Isogen (NIPPON GENE) according to the manufacturer’s instructions. For RNA isolation from exosomes, the pellets were homogenized in 750 µl of Isogen. Then 200 µl of chloroform was added to the sample and the mixed solution was centrifuged. After an additional chloroform extraction and precipitation with isopropanol, the RNA sample was suspended in 20 µl of nuclease-free water.
Microarray analysis
To compare the expression levels ofmiRNA derived from the exosomes isolated by ultracentrifugation and Exoquick, 100 ng of total RNA was labeled and hybridized using Human microRNA Microarray Kit (Agilent Technologies) according to manufacturer’s protocol (Protocol for use with Agilent microRNA microarrays Version 1.5). Hybridization signals were detected with DNA microarray scanner (Agilent Technologies), and the scanned images were analyzed using Agilent feature extraction software.The data discussed in this manuscript have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE35256.
Time-lapse imaging for the transfer of extracellular miRNA
HUVECs were seeded onto35-mm glass-bottom dishesat a density of 5 × 103 cells in EBM. After incubation for 24 h, the exosomes derived from K562/Cy3-miR-92a cells were added into HUVEC mono-culture, and time-lapse images (fluorescence and phase-contrast) were taken by BIOZERO BZ-8000 (KEYENCE, Osaka, Japan) at 15-min intervals for 24 h equipped with an incubation chamber under regulation at 37°C in 5% CO2 humidified atmosphere (Tokai Hit, Fujinomiya, Japan).
Supporting Figure Legends
Figure S1Profiling of extracellular miRNAs in the mono-culture medium vs the co-culture using TaqMan Low-Density Array (TLDA) for human miRNAs. Of the 381miRNAs represented on the card A, 130 miRNAs were detected with Ct values of <36. After the selection of expression level with fourfold greater or lower than that mono-culture medium, the scatter plot showed 74 down-regulated miRNAs (red circle) and 21 up-regulated miRNAs in co-culture medium. These results suggest that the existence of HUVECsglobally leads the decrease of extracellular miRNA in culture medium.
Figure S2The transfer of exosomal miRNAisolated by ultracentrifugation into HUVECs. K562/Cy3-miR-92a cells were cultured in serum-free AIM V medium for 24 h, and the exosomal fractions were isolated by ultracentrifugation. When the exosome fractions isolated by ultracentrifugation were added to HUVEC mono-culture, the signals of Cy3-miR-92a (red) were detected in the cytoplasm of HUVECs.Nuclear counterstaining was performed using DAPI (blue). The size bar indicates 100 µm.
Figure S3Comparison of normalized signal intensities of various miRNAs in the exosomes isolated by Exoquick or ultracentrifugation by miRNA microarray. The X-axis represents the isolation by Exoquick, and the Y-axis represents the isolation by ultracentrifugation. There were no differences in miRNA populations contained in exosomes isolated by the two collecting methods.