Antibodies and inhibitors
The antibodies used targeted the following proteins (dilutions used are included):
GAPDH (CW0101: immunoblotting, 1:1000) from CWBIOTECH; F-actin (40734ES75: immunofluorescence, 1:100) from YEASEN; SNRPA (10212: immunoblotting, 1:500) from Proteintech; CD63 (67605-1-Ig: immunoblotting, 1:5000) from Proteintech; CD9 (60232-1-Ig: immunoblotting, 1:5000) from Proteintech; TSG101 (14497-1-AP: immunoblotting, 1:1000) from Proteintech; GPX4 (67763-1-Ig: immunoblotting, 1:5000; IHC, 1:1000) from Proteintech; and DHODH (14877-1-AP: immunoblotting, 1:2000; IHC, 1:100) from Proteintech.
The inhibitors used are as follows:
Ferrostatin-1 (HY-100579: 60 nM for the in vitro assay) was obtained from MedChem Express, and ferrostatin-1 (HY-100579: 5 mg/kg, intraperitoneal injection for the in vivo assay) was obtained from MedChem Express.
Cell lines and culture
The HepG-2 and HEK-293T cell lines were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). These cell lines were authenticated by the analysis of short tandem repeat (STR) profiles, and all of them matched those of the standard cell lines in the DSMZ data bank. These cells tested negative for cross-contamination of other human cells and mycoplasma contamination. HepG-2 and HEK-293T cells were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin-streptomycin.
Transmission electron microscope assay
Cells were collected and fixed with 2.5% glutaraldehyde. Subsequently, cells were postfixed in 2% tetroxide and dehydrated through a series of gradient ethanol solutions. Samples were embedded in epoxy resin, cut into thin slices, and placed onto a nickel grid. Images were acquired using a Tecnai G2 Spirit transmission electron microscope (Thermo Fisher).
Intracellular ROS measurements
A lipid ROS assay was performed as described previously. Briefly, cells were incubated with PBS containing 10 µM DCFDA dye in a cell culture incubator for 30 min. Cells were then collected and washed twice with PBS followed by resuspension in 200 µl of PBS. ROS levels were analyzed using a Beckman CytoFLEX system through the FITC channel.
Detection of malondialdehyde (MDA)
Analysis of lipid peroxidation was assessed by quantifying the MDA concentration in cell lysates using a Lipid Peroxidation MDA Assay Kit (S0131, Beyotime) in accordance with the manufacturer’s instructions.
Six-week-old male nude mice were used. All animal experiments were carried out under protocols approved by the Animal Care and Use Committee of Fourth Military Medical University.
For in vivo tracking of exosomes, purified exosomes with the indicated modifications were labeled with the fluorescent dye DiR at a final concentration of 8 µM (Invitrogen). Labeled exosomes were collected by ultracentrifugation after washing with saline and stored in saline before use. Mice were injected with labeled exosomes (100 µg at the protein level in 100 µL) via tail vein injection. Mice were subjected to fluorescent living imaging 6 h after injection with an in vivo imaging system (IVIS lumina II).
For orthotopic implantation, six-week-old male nude mice were anesthetized with 3% (w/v) pentobarbital sodium by intraperitoneal injection. Then, 2×106 sorafenib-resistant HepG-2 cells stably expressing luciferase were surgically implanted into the left liver lobes of mice. Tumor growth was monitored by bioluminescence with an in vivo imaging system (IVIS lumina II). Two weeks after inoculation, mice were randomized to each group and began to receive different treatments. In the sorafenib treatment group, sorafenib (30 mg/kg) was given every 3 days. In the sorafenib and exosome combination treatment group, sorafenib was administered at the same dose, and 100 µg of the indicated exosomes (at the protein level) was injected via the tail vein 24 h after every sorafenib administration.
This experiment was performed as previously described. Briefly, sections (4 µm thick) of paraffin-embedded samples were deparaffinized and rehydrated in a graded series of ethanol. After inactivation of endogenous peroxidase activity with 3% H2O2 in methanol for 10 min, the sections were washed three times in PBS and blocked with goat serum for 20 min. Then, they were incubated with primary antibodies in a humid container at 4°C overnight. After the addition of the PowerVisionTM complex, tissue sections were incubated at 37°C for 20 min followed by treatment with DAB. PBS in place of primary antibody was used as a negative control.
The data are presented as the mean±s.e.m. from at least three independent experiments. Statistical analysis was performed using GraphPad Prism software. A random number table was used to randomize the mice into control and treatment groups. The numbers of mice were determined on the basis of our pretests and previous experience with similar experiments. A value of P<0.05 was considered statistically significant. The statistical tests were two-sided.