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All human-related experimental protocols were given consent by the Institutional Review Board, approved by the appropriate institutional review committees (ECSHU2020-004), and performed in accordance with the Helsinki Declaration as revised in 2013. Three pooled serum samples (n = 3 samples/pool) were collected from healthy males aged 50–60 years. Serum pools were obtained as follows. Blood without anticoagulant was incubated at room temperature for 1 h and centrifuged at 2,500 ×g for 15 min at 4 °C. The supernatant was then collected. Three samples were collected into one pool. EV isolation was conducted using ExoQuickTM Exosome Precipitation Solution (EXOQ5A-1, System Biosciences, USA) according to the manufacturer’s instructions. In brief, 250 μL of the pooled serum samples was added to the appropriate volume of ExoQuick precipitation solution. The solution was mixed well, refrigerated for 30 min at 4 °C, and then centrifuged at 1,500 ×g for 30 min. After centrifugation, the EV pellets at the bottom of the vessel were collected and resuspended in 100 μL of sterile phosphate buffer solution (PBS). The EVs were used immediately or stored at −80 °C for further study. A total of 10 μL of the isolated EVs was dropped onto an ultra-thin carbon film for transmission electron microscopy (TEM) imaging. The liquid was removed using filter paper, and the film was washed thrice with distilled water. The carbon film was dried naturally at room temperature. TEM images were obtained using an LVEM5 transmission electron microscope (Delong Instruments) operated at 5 kV.
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The EV pellets were resuspended and diluted with the appropriate volume of PBS. Next, the concentration and size distribution of EVs were determined by nanoparticle tracking analysis (NTA, Version 3.1 Build 3.1.54, Malvern, UK) as previously described[39]. The EV experiments were adjusted to the MISEV2018 guideline[40].
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Human alveolar epithelial A549 cells were acquired from the Cell Bank (Chinese Academy of Sciences) and cultured with Dulbecco’s modified Eagle’s medium (DMEM, Corning, USA) plus 1% penicillin/streptomycin and 10% fetal bovine serum (BioInd, Israel) at 37 °C and 5% CO2. PM2.5 was purchased from NIST (SRM 1650b, USA) and dissolved in an appropriate volume of dimethyl sulfoxide (DMSO). Prior to treatment, the A549 cells were seeded in 12-well plates at a density of 2 × 105 cells/mL and allowed to attach for 12 h. The cells were then exposed to 50 μg/mL PM2.5 with or without 10 μg/mL EV pre-treatment for 24 h. An equal volume of DMSO in DMEM (DMSO < 0.1%) was used as the negative control, and an equal volume of sterile 1× PBS was used as the EV control. The concentration of PM2.5 utilized in the experiments was based on a previous study[41]. According to the Stochastic Human Exposure and Dose Simulation (SHEDS-PM) model[42], the exposure dose of cells used in this work is parallel to that of real-life exposure.
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Cells were incubated in 96- or 6-well plates and treated with 10 μg/mL EVs or PBS for 24 h. Next, the cells were exposed to PM2.5 for 24 h and subjected to cell viability and apoptosis analyses. An enhanced Cell Counting Kit-8 (CCK-8; Beyotime, China) was used for the cell viability test. Briefly, 10 μL of enhanced CCK-8 solution was added to each well, and the plate was incubated for 2 h according to the manufacturer’s instructions. Cells were subjected to flow cytometric analysis with an Annexin V-FITC/PI kit (Dojindo, #AD10, Japan) according to the manufacturer’s instructions to determine cell apoptosis. A549 cells in 1× binding buffer were incubated in the dark with FITC Annexin V and PI for 15 min at room temperature and then immediately analyzed by flow cytometry.
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The supernatants (without EVs) and EV pellets were lysed with EV-specific lysis buffer (Umibio, Shanghai, China). A549 cells were lysed with RIPA (KeyGen Biotech, China) containing 1% PMSF and phosphatase inhibitor cocktail (1 tablet per 10 mL of lysis buffer, 4906837001-PhosSTOP™, Roche). Western blot analysis was performed as previously described[39], and β-actin was used as an internal control. Primary antibodies against β-actin were purchased from Bioworld Technology, Inc. (USA). Primary antibodies against Bax, Bcl2, caspase 3, CD63, CD9, and Tsg101 were purchased from ABclonal (China). Primary antibodies against p-AKTThr308 and p-AKTSer473 were purchased from Cell Signaling Technology (USA). Primary antibodies against AKT were purchased from ProteinTech (China). All of the primary antibodies used in this study were diluted to 1:1,000 with 5% BSA, and all of the secondary antibodies were diluted to 1:10,000 with 5% defatted milk. The corresponding Research Resource Identifiers are reported in Supplementary Table S1, available in www.besjournal.com.
Antigen Description of immunogen Source, host species, catalog No.,
clone or lot No., RRIDConcentration used Anti-CD63 Recombinant fusion protein containing a sequencecorresponding to amino acids
103-203 of human CD63 (NP_001771.1)ABclonal Technology, rabbit monoclonal antibody, A5271, RRID: AB_2766092 1:1,000 in 5% BSA (WB) Anti-CD9 Recombinant protein of human CD9 ABclonal Technology, rabbit monoclonal antibody, A10789, RRID: AB_2758224 1:1,000 in 5% BSA (WB) Anti-Tsg101 A synthetic peptide corresponding to a sequence within amino acids 300 to the
C-terminus of human TSG101 (NP_006283.1)ABclonal Technology, rabbit monoclonal antibody, A2216, RRID: AB_2764231 1:1,000 in 5% BSA (WB) Anti-Bax A synthetic peptide corresponding to a sequence within amino acids 1-100 of human BAX (NP_620116.1) ABclonal Technology, rabbit monoclonal antibody, A12009, RRID: No 1:1,000 in 5% BSA (WB) Anti-Bcl2 A synthetic peptide corresponding to a sequence within amino acids 1-100 of human Bcl-2 (NP_000624.2) ABclonal Technology, rabbit monoclonal antibody, A11025, RRID: AB_2758373 1:1,000 in 5% BSA (WB) Anti-caspase 3 Recombinant fusion protein containing a sequence corresponding to amino acids
55-160 of human Caspase-3 (NP_004337.2)ABclonal Technology, rabbit monoclonal antibody, A21156, RRID: No 1:1,000 in 5% BSA (WB) Anti-AKTThr308 Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues around Thr308 of mouse Akt. Cell Signaling Technology, rabbit monoclonal antibody, # 2965, RRID: AB_2255933 1:1,000 in 5% BSA (WB) Anti-AKT AKT fusion protein Ag0213 Proteintech,rabbit monoclonal antibody, 10176-2-AP, RRID: AB_2224574 1:1,000 in 5% BSA (WB) Anti-β actin Recombinant full length Human β-Actin. Bioworld,mouse monoclonal antibody, BS6007M, RRID: No 1:1,000 in 5% BSA (WB) Goat anti-mouse IgG Mouse IgG (H+L) Bioworld,goat, BS12478, RRID: AB_2773727 1:10,000 in 5% milk (WB) Goat Anti-Rabbit IgG Peroxidase-conjugated Affinipure Goat
Anti-Rabbit IgG (H+L)Jackson ImmunoResearch Labs, goat,
111-035-003, RRID: AB_23135671:10,000 in 5% milk (WB) Table S1. Primary antibodies used
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All experiments were performed independently at least three times. All data are presented as mean ± SD and analyzed using SPSS (version 20). One-way ANOVA followed by Bonferroni’s post hoc test was performed for multiple group comparisons. A P-value less than 0.05 was considered statistically significant.
Human Serum-derived Extracellular Vesicles Protect A549 from PM2.5-induced Cell Apoptosis
doi: 10.3967/bes2021.006
- Received Date: 2020-09-04
- Accepted Date: 2020-11-23
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Key words:
- Cell apoptosis /
- PM2.5 /
- Extracellular vesicles /
- Therapy /
- AKT
Abstract:
Citation: | ZHOU Qiu Lian, BAI Yu Zheng, GAO Juan, DUAN Yi, LYU Yi Cheng, GUAN Long Fei, ELKIN Kenneth, XIE Yu Ling, JIAO Zheng, WANG Hong Yun. Human Serum-derived Extracellular Vesicles Protect A549 from PM2.5-induced Cell Apoptosis[J]. Biomedical and Environmental Sciences, 2021, 34(1): 40-49. doi: 10.3967/bes2021.006 |