Alpha mouse liver 12 (AML12) cells and HEK293T cells had been cultured in excessive glucose DMEM medium (Logan, Utah, U.S.A.) containing 10% exosome-free FBS (fetal bovine serum), 1% L-glutamine and 1% penicillin–streptomycin (Logan, Utah, U.S.A.) in a humidified environment at 37 °C with 5% CO2. The exosome depleted FBS was obtained by eradicating exosome with ultracentrifugation at 120,000g for 3 h at 4 °C (Beckman Coulter X-90 centrifuge, SW41 Ti rotor).
AML12 cells had been transfected with scramble siRNA and siRNA goal genes of curiosity by utilizing HiGene transfection reagent (C1506, Applygen Expertise Inc.) based on producer’s instruction. The designed sequences of si-NC, si-Rab4, si-Rab22a, si-Rab11a si-Rab35, si-Rab9, si-Nsf, si-Vps39, si-Vps18, si-Rab33b, si-Rab24, si-Tfeb and si-Rab14 (GenePharma) had been listed in Further file 1: Desk S1.
Plasmid development, lentivirus package deal and an infection
Ldlr coding areas had been cloned into pWPI vector changing the IRES-EGFP as described beforehand . The Ldlr expressing vector was transfected into HEK293T cells along with psPAX2 and pMD2G on the molar ratio of 4:3:1 with HighGene transfection reagent (ABclonal). Lentivirus particles had been harvested from the supernatant filtered by 0.45 μm filters 72 h after transfection and saved at − 80 °C. For an infection, AML12 cells cultured in plates had been incubated with the lentivirus on the MOI of 200 within the presence of polybrene (8 μg/ml).
Pink cell membrane particles preparation
Complete blood samples had been collected from the orbit of male mice (C57BL/6) aged 6–8 weeks with the addition of 1.5 mg of EDTA for anticoagulation goal. Blood samples had been centrifuged at 3000 rpm/min for 10 min at 4 °C to take away the plasma and picked up RBCs had been washed with pre-cooled 1× PBS for 5 occasions. Then, 0.1× PBS (PBS: deionized H2O = 1:9) was added and positioned at 4 °C for two h for hemolysis. The launched hemoglobin was eliminated by centrifugation at 12,000 rpm for 15 min, and the pellet was collected and washed for five occasions till the pellet turned gentle pink shade. The crimson cell membrane pellets had been re-suspended with a hydration resolution consisted of 1.8 ml of 1× PBS and 200 μl glycerol, adopted by homogenization 10 min at 10,000 rpm with a miniature high-speed dispersing homogenizer (F6/10, jingxin expertise, shanghai) underneath ice water bathtub.
Labeling of RCMPs and monitoring of mobile uptake
The RCMPs had been incubated with DiI at 37 °C for 20 min in darkish, and switch to 4 °C for 10 min, then centrifuged at 4 °C for 12,000g for 15 min to take away the unbound dye. The labeled RCMPs had been resuspend in PBS prior to make use of. AML12 cells had been incubated with DiI -labeled RCMPs for six h. Then, the medium was eliminated and AML12 cells had been washed twice with PBS. Mobile internalization of DiI-labeled RCMPs had been analyzed by laser scanning confocal microscope or circulation cytometer (Beckman CytoFLEX).
Exosome isolation and characterization
AML12 cells with transfection/an infection had been cultured in DMEM medium. For supplementation of RCMPs, cells had been added with RCMPs (80 μg/ml) and incubation for six h earlier than swap to exosome-free medium for extra tradition of 48 h. Cells had been discarded by centrifugation at 500g for 10 min and the residual mobile particles had been eliminated by centrifugation at 5000g for 20 min. The collected supernatants had been filtered by 0.22 μm filters, after which had been ultracentrifuged at 100,000g for two h (Beckman Coulter X-90 centrifuge, SW41 Ti rotor). The remoted exosomes had been resuspended in 1× PBS and saved at − 80 °C until use. Measurement distribution and focus of exosomes had been analyzed by ZetaView® instrument (Particle Metrix, USA). The samples had been loaded into the pattern chamber at ambient temperature. Then, the focus was calculated based on the dilution fold.
For transmission electron microscopy evaluation of the exosome morphology, remoted exosomes had been allowed to be mounted for six h in 2.5% glutaraldehyde in phosphate buffer at 4 °C. Then the samples had been dried on a copper grid 5 min, adopted by rapid statement at JEM-2000EX electron microscopic evaluation (HITACHI, HT7800/HT7700).
Electron microscopy and MVB quantification
AML12 cells transfected with si-NC and si-Rab4 had been washed with PBS and glued with 4% paraformaldehyde at room temperature. Then, the cells had been added onto grid, stained (2% uranyl acetate) and imaged by electron microscopy (HT7800, Hitachi). MVBs numbers per profile had been calculated.
For immunofluorescence assay, AML12 cells with indicated therapies had been cultured in confocal dish (35 mm) and incubated for 48 h. Then, the tradition medium was discarded and the cells had been washed with PBS, adopted by repair with 4% paraformaldehyde for 20 min. Then, cells had been stained with major antibody (anti-HRS, sc-271455) in a single day, adopted by secondary antibody [Goat anti-mice 633, Invitrogen, A-21050)] at room temperature for 1 h at midnight. Lastly, the nuclei had been stained with Hoechst (1:1000). Pictures had been processed utilizing laser scanning confocal microscope. HRS spots per cell had been calculated utilizing Picture J.
In vitro and in vivo monitoring of exosomes
Exosomes had been labelled with DiR/DiI by direct incubation with the dye (1 μM in last focus, Invitrogen, China) at 37 °C for 10 min, after which the free dye was eliminated by centrifugation at 12,000 rpm for 10 min, and the precipitate was re-suspended with PBS.
For in vitro experiment, AML12 cells had been seeded into confocal dish and incubated with DiI-labeled exosomes (last focus of 40 μg/ml) for six h. Cells had been then washed with PBS and glued in 4% paraformaldehyde for 10 min at room temperature. The nuclei had been stained with Hoechst (C1022, Beyotime, China) for 10 min at room temperature, adopted by PBS 3 times. Mobile uptake of exosomes in vitro was noticed by confocal microscopy (Nikon, Tokyo, Japan). The entire experiment was stored in darkish.
For in vivo tracing, mice had been intravenously injected with freshly ready DiR or DiI-labeled exosomes samples. After 6 h, the DiR fluorescence sign of the entire mouse and main organs (coronary heart, liver spleen, lung and kidney) had been imaged by the in vivo imaging system (IVIS, PerkinElmer, Thermo Fisher, USA), and the DiI fluorescence sign was imaged by confocal microscopy on the tissue sections.
Protein lysis from cells, exosomes and tissues had been ready and the protein focus was decided by Pierce BCA Protein Assay Equipment (Thermo Fisher Scientific, Waltham, USA.). Protein Samples had been separated by 10% or 12% SDS-PAGE gels and transferred to nitrocellulose filter membranes. The nitrocellulose filter membranes had been blocked with 3% skim milk in tris buffered saline (TBS) containing 0.1% Tween-20 (TBST) at 4 °C in a single day, after which incubated with major antibodies adopted by a horseradish peroxidase-conjugated secondary antibodies (washed with TBST 3 times earlier than every operation, 5 min every time). Major antibodies used had been anti-LDLR (10785-1-AP, Proteintech), anti-GM130 (sc71166, Santa Cruz), anti-TSG101 (ab83, Abcam), anti-CD63 (ab134045, Abcam), anti-GAPDH (60004-1-lg, Proteintech), secondary antibodies used had been anti-Rabbit (7074, CST) and anti-Mouse (7076, CST).
Reverse transcription and quantitative polymerase chain response
The collected RNA of cells and exosomes had been extracted utilizing TRIzol reagent (Invitrogen, USA), and complementary DNA (cDNA) was obtained by Transcriptor Reverse Transcriptase (Indianapolis, USA) based on producers’ directions. qPCR reactions (in 20 μL system) had been carried out by FastStart Important DNA Inexperienced Grasp (Roche, Basel, Switzerland). The expression of goal gene at RNA ranges was normalized to Gapdh for comparability and calculated utilizing the two−∆∆Ct. For evaluation of siRab4 abundance in exosomes, RNA was remoted and reverse transcribed with miRNA transcriptase package. U6 served as inner management. All PCR reactions had been carried out in triplicates. The primer sequences had been utilized in Further file 1: Desk S2.
Exosome therapy in Ldlr
All animal experiments had been authorised by the Animal Care and Use Committee of Air Pressure Medical College. Animal experiments had been carried out conforming to the Directive 2010/63/EU of the European Parliament. Ldlr−/− mice (C57BL/6 background) had been bought from the Mannequin Animal Analysis Middle of Nanjing College. All mice had been fed with high-fat weight loss program for 8 weeks after which handled with indicated exosomes by way of tail vein injection on the dose of 4 μg/g physique weight as soon as per week for 8 weeks. After 8 weeks of exosomes intervention, all mice had been intraperitoneally injected with 1% pentobarbital sodium at 0.1 ml/10 g, after which had been killed by cervical dislocation. The primary tissues (coronary heart, liver, spleen, lung, kidney and aortic) had been separated for subsequent evaluation. Blood samples had been collected from mice after in a single day fasting. All samples had been allowed to face at room temperature for two h then centrifuged at 4 °C for 3000g for 15 min. The collected supernatants had been assayed for AST, ALT, complete triglyceride, complete ldl cholesterol, LDL ldl cholesterol and HDL ldl cholesterol (Wuhan Servicebio Expertise CO, LTD). For histological research, the primary organs (coronary heart, liver, spleen, lung and kidney) had been rigorously harvested and sectioned for H&E staining. Aorta, aortic roots and liver sections had been additional stained with Oil-red-O for lipid deposition evaluation. The physique weights of the mice had been recorded weekly for 8 weeks.
Information are expressed as imply ± SEM. A method ANOVA and t take a look at had been used for distinction comparability by GraphPad prism 9.0. P values < 0.05 had been thought-about statistically important.