Cells have not undergone any special treatment. All exosome preparation were derived from culture supernatant after cells have been cultured in serum-free medium for 48h-72h. Culture supernatant was collected and cleared (2x10min, 500g, 1x20min, 2000g, 1x30min, 10000g), filter sterizied (0.2µm) and were centrifuged (120min, 100000g). The pellet was washed (PBS, 120min, 100000g), resuspended in 40% sucrose overlaid by a discontinuous sucrose gradient (30%-5%) and centrifuged (16h, 100000g). Exosomes were collected from the 10%-5% sucrose interface (light density fractions, d: 1.15-1.56g/ml).
Growth protocol
The human pancreatic adenocarcinoma lines lines A818.4 and Capan-1 are maintained in RPMI1640/10%FCS/pyruvate/L-glutamine/antibiotics. Confluent cells are trypsinized and split. A818.4-Tspan8kd, A818.4-CD151kd, A818.4-CD44v6kd, Capan-1-CD44v6kd cells were derived from transfection with shRNA (Qiagen, Hilden, Germany) and maintained in RPMI1640/10%FCS/pyruvate/L-glutamine/antibiotics containing 0.5µg/ml G418. Single cell kd clones were derived by 2 rounds of single cell cloning. The human colorectal cancer lines HT29 and SW948 are maintained in RPMI1640/10%FCS/L-glutamine/antibiotics. Confluent cells are trypsinized and split. Ht29-cld7 kd, SW948-cld7 kd and SW948-CD44v6 kd lines were derived from transfection with shRNA (Qiagen, Hilden, Germany) and maintained in RPMI1640/10%FCS/pyruvate/L-glutamine/antibiotics containing 0.5µg/ml G418. Single cell kd clones were derived by 2 rounds of single cell cloning.
Extracted molecule
total RNA
Extraction protocol
Cell and exosome mRNA/microRNA was extracted using the miRNeasyMinikit following the supplier's suggestion (Qiagen, Hildesheim, Germany). Exosomes were pretreated with RNAse to avoid contamination by attached free mRNA.
Label
Cy3
Label protocol
Labeling was done as described in miRNA Microarray System protocol v 1.7 from Agilent Technologies
Hybridization protocol
Hybridization was done as described in miRNA Microarray System protocol v 1.7 from Agilent Technologies
Scan protocol
Scanning was done on Agilent Microarray Scanner G2565C
Description
miRNA HCX_03
Data processing
IPA was used for miRNA analysis and for correlating miRNA with protein expression after predicted targets were selected by miRNA databases (http://www.microrna.org,http://www.targetscan.org). For pathway analysis PANTHER (http://pantherdb.org), KEGG (http://www.kegg.jp), Reactome (https://reactome.org), and STRING (http://string-db.org) databases were used after normalization, data were compared between identical non-transfected versus transfected cells and exosomes, respectively. In most instances, 2-fold differences were considered significant. In exosome preparations, 1.5-fold differences were occassionally included. For processed data, the programm suggestions were followed.
