Cells and culture
Human lung carcinoma cell line A549 (RCB0098) and human colon carcinoma cell lines HCT116 (RCB2979) and COLO205 (RCB2127) were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan. A549 and HCT116 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM). COLO205 cells were cultured in RPMI 1640 medium. The cells were maintained in their respective culture media supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin at 37 °C in 5% CO2 and 95% air.
Exosomes were prepared by the standard ultracentrifugation method according to a previous report , and this method was performed as we reported previously . In brief, A549 and HCT116 cells (3 × 106 cells) were seeded in a 100-mm dish in culture medium with 10% FBS. After 24 h, culture medium was changed to culture medium without FBS and incubated. COLO205 cells (6 × 106 cells) were seeded in a 100-mm dish in culture medium without 10% FBS and incubated. The culture media were collected after 72 h incubation and the exosomes were isolated by the following three methods: ultracentrifugation, ExoQuick-TC® (System Biosciences Inc., Palo Alto, CA, USA) and Total Exosome Isolation® (Thermo Fisher Scientific Inc., Waltham, MA, USA). The ultracentrifugation method was performed as follows. The collected medium was centrifuged at 2000 g for 30 min, and then at 10,000 g for 30 min to remove cell debris. The supernatant was centrifuged at 100,000 g for 70 min to purify exosomes. The pellet was washed with PBS and ultracentrifuged at 100,000 g for 70 min again. The pellet was resuspended with PBS and stored until use. Exosome isolation using ExoQuick-TC and Total Exosome Isolation was performed according to the manufacturer’s instructions. In brief, the collected medium was centrifuged at 2000 g for 30 min and supernatant was collected. One-fifth of ExoQuick-TC Exosome Precipitation Solution or half of Total Exosome Isolation were added to the supernatant and their suspension was incubated overnight at 4 °C. The suspension was centrifuged at 1500 g for 30 min for ExoQuick-TC or at 10,000 g for 60 min for Total Exosome Isolation. The pellet was resuspended with PBS. Exosome protein content was qualified using the BCA protein assay kit (Thermo Fisher Scientific) before further experiments.
Uptake of DiO-labeled exosomes by recipient cells
Twenty-four μg of exosomes were incubated with lipophilic tracer DiO solution (Thermo Fisher Scientific) for 20 min at 37 °C. Excessive DiO was removed with Exosome Spin Columns (MW 3000) (Thermo Fisher Scientific). Exosome labeling efficiency was analyzed with an Infinite® 200 PRO fluorometer (TECAN, Männedorf, CHE). The cells were seeded in an 8-well chamber slide (1 × 104 or 4 × 104 cells/well) and incubated for 24 h. DiO-labeled exosomes (8 μg) were added to the culture media of the recipient cells and incubated for 3 h at 37 °C. The recipient cells were fixed with 4% paraformaldehyde at room temperature for 10 min and permeabilized with 0.1% Triton X-100 at room temperature for 5 min. The cells were stained with Alexa Fluor 555 phalloidin (Thermo Fisher Scientific) at room temperature for 30 min and mounted in Prolong® Diamond Antifade Reagent with DAPI (Thermo Fisher Scientific), and the slide was covered with cover glass. The cells were visualized with an EVOS FL fluorescence microscope (Thermo Fisher Scientific).
Total RNA extraction from cell lines
Total RNA was extracted from cell pellets using TRIzol reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. In brief, the cells were lysed by TRIzol and chloroform was added to the cell lysis. The suspension was centrifuged at 12,000 g for 15 min and aqueous phase was collected. Isopropyl alcohol was added to the aqueous phase and then was centrifuged at 12,000 g for 10 min. The supernatant was removed and 75% ethanol was added to the pellet for washing RNA. The suspension was centrifuged at 7500 g for 10 min and the supernatant was removed. The pellet was dissolved by RNase-free water. The quantity of total RNA was determined using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).
Quantitative real-time PCR
Total RNA (0.2 μg) from each sample was reverse transcribed to complementary DNA (cDNA) for real-time PCR using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan), according to the manufacturer’s protocol. In brief, the reaction was conducted by incubating for 10 min at 25 °C followed by 60 min at 42 °C and 5 min at 95 °C. PCR reaction was monitored in real-time with a Thermal Cycler Dice Real Time System (TaKaRa Bio, Otsu, Japan). The PCR reaction was carried out in 20 μl of a reaction mixture composed of Thunderbird SYBR qPCR Mix (Toyobo) and 0.5 μM of each primer. The reaction mixture was subjected to an initial denaturation at 95 °C for 20 s, followed by 50 cycles of amplification at 95 °C (3 s) for denaturation, and at 60 °C (30 s) for annealing. After the cycles, a melting curve was checked to confirm the single product. Relative expression levels of target genes were calculated by the delta-delta Ct method with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as a reference gene. The PCR primer sequences used for detecting gene expression of clathrin, caveolin-1 and GAPDH were as follows: clathrin forward 5’-GTTACTGCACCTCATGAAGCC-3′ and reverse 5’-AGTTCTTCAGCACCGGCTAA-3′; caveolin-1 forward 5’-GTCAACCGCGACCCTAAACA-3′ and reverse 5’-GATGCCAAAGAGGGCAGACA-3′; GAPDH forward 5’-TTCTTTTGCGTCGCCAGCCGA-3′ and reverse 5’-GTGACCAGGCGCCCAATACGA-3′.
Whole-cell protein extracts
Cells were lysed with RIPA buffer (Wako, Osaka, Japan), composed of 50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.5% SDC, 0.1% SDS, 150 mM NaCl, and protease inhibitor cocktail (Nacalai tesque, Kyoto, Japan). The supernatants obtained after centrifugation at 15,000 g for 10 min at 4 °C were used as whole-cell protein extracts.
Total exosome protein (2 μg) was resuspended by 5× RIPA buffer (125 mM Tris-HCl, 750 mM NaCl, and 5% NP-40, 5% sodium deoxycholate and 0.5% SDS) and then suspension was sonicated for 5 min and incubated for 15 min on ice. The suspension of whole-cell protein extracts (20 μg) was boiled in a sixth-volume of sample buffer (Nacalai tesque) and separated on 12% SDS-polyacrylamide gels. Proteins on the gels were transferred to polyvinylidene difluoride membranes, which were blocked with Blocking One (Nacalai tesque) overnight at 4 °C. Anti-CD63 rabbit polyclonal antibody (1:200; System Biosciences Inc.), anti-CD9 rabbit polyclonal antibody (1:200; System Biosciences Inc.), anti-CD81 rabbit polyclonal antibody (1:200; System Biosciences Inc.), anti-HSP70 rabbit polyclonal antibody (1:500; System Biosciences Inc.), anti-clathrin mouse polyclonal antibody (1:2000; BD Biosciences, Franklin Lakes, NJ, USA), anti-caveolin-1 mouse monoclonal antibody (1:500; BD Biosciences) and anti-β-actin rabbit monoclonal antibody (1:5,000; Cell Signaling Technology, Danvers, MA, USA) were used as primary antibodies. The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The secondary anti-rabbit IgG or the secondary anti-mouse IgG was diluted to 1:5,000–1:10,000 or 1:5,000, respectively. Protein/antibody complexes were visualized with Chemi-Lumi One Super (Nacalai tesque) and detected using an Image Quant LAS 4000 (GE Healthcare Biosciences, Piscataway, NJ, USA).