Cell Culture and Infection
A549 adenocarcinoma cells were purchased from the Cell Resource Center of the Shanghai Academy of Sciences, Chinese Academy of Sciences.
Human lung epithelial A549 cells were cultured in F-12K Nutrient Mixture (GIBCO, Grand Island, NY, USA) at pH 7.2, supplemented with 10% fetal bovine serum (GIBCO) and penicillin (100 U/mL)/streptomycin (100 µg/mL) and grown in a cell culture incubatorat 37 ℃, under conditions of5%CO2 and saturatedhumidity. A549 cells (80% confluent) were infected with H5N1 and H9N2 viruses atamultiplicity of infection(MOI)of 1 for 1 h, and culture medium was added at 24 h.
Virus Tissue Culture Infective Dose (TCID)
A/Chicken/Jiangsu/07/2002 (H9N2), and A/Chicken/Henan/12/2004 (H5N1) were obtained from the Wuhan Institute of Virology, Chinese Academy of Sciences.The virus was diluted 10-fold with a virus dilution solution (phosphate buffer saline+0.1%BSA) , and 100 μLvirus solution was added to cells for 1 h of incubated in a cell culture incubator at 37 ℃, with 5% CO2 and saturated humidity. After the virus and cells were incubated for 1h, the virus–serum mixture was replaced with 5 mL of viral growth medium (serum-free complete F-12K supplemented with 2 µg/mL of TPCK-treated trypsin). After culture for 24 h , the median TCID (TCID50) was calculated by the method of Reed and Muench .From the of TCID50value, we calculated the MOI by the following formula.
Extraction of Mitochondrial Proteins from Virus Infection
Virus-infected cells were washed once with precooled PBS, and lysis buffer (25 mM mannitol, 0.5 mM EGTA, 5 mM HEPES, 0.1% BSA [w/v]) was added. The cells were scraped and placed in a homogenizer for homogenization. The cell sample was centrifuged at 600 ×g for 5 min at 4 °C, and the supernatant was collected.The supernatant obtained by centrifugation in the previous step was centrifuged at 10,300×g for 10 min at 4 ℃, and the precipitate was mitochondrial protein. Centrifugation was repeated once, and the precipitate was combined with that obtained in the previous step to further maximally enrich mitochondrial protein. The obtained mitochondrial protein was suspended in acetone precooled to -20 ℃, stored at -20 ℃for 12 h or more, and centrifuged at 8750 ×g for 35 min at 4 ℃. The supernatant was then removed, and the centrifugation step was repeated once. On an ultraclean work bench, samples were naturally air-dried on ice, and an appropriate amount of lysate was added to fully dissolve the precipitate. Protein was further dissolved by vortexing[18-19].
The sample was collected and the protein concentration was determined using a bicinchoninic acid(BCA) protein assay kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. The samples were then aliquoted and stored at −80 ℃ until subsequent use.
Two-Dimensional (2-D) Gel Electrophoresis
Rehydration solution (8 M urea; 2 M thiourea; 4.0% [w/v] CHAPS; 20 mM Tris base; 20 mM DL-dithiothreitol; 0.5% [v/v]pH 3-10 amidine, and 10% [v/v] bromophenol blue) was added to the protein sample. For isolation of proteins by isoelectric focusing(IEF), a salt bridge was formed at the two poles of the electrophoresis tank, the supernatant of the centrifuged protein sample was added uniformly to the electrophoresis tank, and the isoelectric focusing strip was removed from the -20 ℃freezer (17 cm, pH 3-10) and equilibrated to room temperature. The sample was added to the electrophoresis channel, and the appropriate amount of mineral oil was added to cover the strip. The program was set as follows: 50 V for 14 h, passive rehydration; 500 V for 1 h, linear; 1000 V for 1 h, rapid; 5000 V for 1 h, rapid; 8000 V for 1 h, linear; and rapid ramping to 8000 V for 60,000 Vh. After isoelectric focusing, strips were equilibrated in equilibration buffer (6 M urea, 20% glycerol, 2% SDS, 25 mM Tris-HCl [pH 8.8]) containing 0.2% (w/v) dithiothreitol for 15 min and then in the same buffer containing 3.0% (w/v) iodoacetamide and 0.175% (v/v) bromophenol blue for 15 min. Separation in the second direction was performed via 12.5% SDS-PAGE under a constant current of 25 mA, and gels were stained with Coomassie Brilliant Blue G-250. After decolorization, analysis was performed using ImageMaster software to match gel spots, and gray values that were significantly different (gray value ≥ 2.0-fold) between the H9N2-infected and H5N1-infected groups were selected for MS analysis.
In-gel Trypsin Digestion, MS and Data Searching
The sample was mixed in an equal ratio with 10 mg/mL α-cyano-4-hydroxcinnamic acid, directly spotted onto a spotting plate, and allowed to dry at room temperature. Peptide mass spectra were obtained with a 5800 MALDI TOF/TOF mass spectrometer (AB SCIEX, Foster City, USA). The MS/MS data of the peptide mass fingerprint (PMF) were submitted to the online software Mascot (Matrix Science, Boston, MA, USA) for identification according to the NCBIProt database. The parameters were as follows: taxonomy, Homo sapiens; enzyme, trypsin; allowed missed cleavages, two; variable modification, oxidation (M); fixed modification, carbamidomethylation (C); and number of peptide charges, +1. The peptide and fragment mass tolerances were set at ±1.2 Da and ±0.6 Da, respectively. The data format was selected as Mascot generic, and the instrument was selected as MALDI-TOF-TOF. Proteins with a score greater than 30were regarded as trustworthy proteins..
Western Blot Analysis
The extracted total cell lysate and mitochondrial proteins of H5N1 and H9N2 virus-infected A549 cells were quantified with a BCA kit (Sangon Biotech, Shanghai, China). Mitochondrial proteins (40 µg) and total cell lysate (40 µg) were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (BBI Life Sciences). After blocking with 5% (w/v) skim milk in TBST (50 mM Tris[pH 8.0], 150 mM NaCl, 0.1% [v/v] Tween-20) for 1 h at 37 ℃, membranes were incubated separately overnight at 4 ℃ with rabbit monoclonal or polyclonal antibodies against ECHS1 (ab170108), MDH2 (ab181873) (Abcam), ATP5F1 (15999-1-ap), HSPA1L (13970-1-ap), BAX (50599-2-Ig), and Caspase 3 (66470-2-Ig) (Proteintech). After three washes with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG (used at a 1:5000 dilution, Proteintech) for 1 h at room temperature and were then washed three times with TBST. The immunoreactive protein bands were detected using enhanced chemiluminescence reagent (ECL; Advansta, CA, USA), with TOM40 (18409-1-ap) (Proteintech) and β-actin (66009-1-Ig) (Proteintech) as the loading controls.
Use the Omicsbean online analysis software (http://www.omicsbean.cn) was used with the accession number of each identified protein to perform Gene Ontology (GO) classification andKyoto Encyclopedia of Genes and Genomes (KEGG) analysis for signaling pathways. The accession number of each identified proteinwas submitted forSTRING(https://string-db.org)analysis for functional protein association networks construction.Regular functional analysis was performedusing the tools in the Swiss-Prot database (http://uniprot.org)[17,20].
The statistical significance of differences between groups was determinedbyusinga paired, nonparametricStudent’s T-test. P<0.05wasconsidered statistically significant.The experiment was repeated three times.