Cells and viruses
The H1N1 SIV A/swine/Shanghai/3/2014 (SH/2014) strain was isolated in our laboratory from pigs with clinical symptoms of swine influenza. A549 cells were obtained from ATCC (Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) at 37 °C with 5% CO2.
Sample preparation for proteomic analysis, protein isolation and iTRAQ reagent labeling
A549 cells infected with SIV strain SH/2014 at a multiplicity of infection (MOI) of 1 or uninfected cells were collected at 24 h post-infection (h.p.i.). The cells were lysed with 400 µL of lysis buffer (100 mM NH4HCO3 (pH 8), 6 M urea and 0.2% SDS) followed by ultrasonication on ice for 10 min. The supernatant was collected and reduced with 10 mM DTT at 56 °C for 1 h and subsequently alkylated with sufficient iodoacetamide for 1 h. The extracted proteins were precipitated with precooled acetone, washed twice, and dissolved in dissolution buffer containing 0.1 M triethylammonium bicarbonate (TEAB) and 8 M urea (pH 8.5). The protein concentration was quantified with a Bradford protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). 100 μg of each protein sample was digested with Trypsin Gold (Promega, Madison, WI, USA) at 37 °C overnight and then labeled with different iTRAQ tags (iTRAQ® Reagent-8PLEX Multiplex Kit, Sigma–Aldrich, St. Louis, MO, USA). The SIV-infected samples were labeled with iTRAQ 113 (IT113), iTRAQ 114 (IT114), or iTRAQ 115 (IT115), and the mock-infected samples were labeled with iTRAQ 116 (IT116), iTRAQ 117 (IT117), or iTRAQ 118 (118). The labeled samples were then mixed with shaking for 2 h at room temperature (RT). The reaction was stopped by adding 100 μL of 50 mM Tris–HCl (pH = 8). All labeled samples were mixed to be of equal volume, desalted and lyophilized.
The iTRAQ-labeled peptide mixtures were fractionated using a Waters BEH C18 column (5 μm, 4.6 × 250 mm) with a Rigol L3000 HPLC system. The column oven temperature was set to 50 °C. Mobile phase A (2% acetonitrile adjusted to pH 10.0 using ammonium hydroxide) and mobile phase B (98% acetonitrile adjusted to pH 10.0 using ammonium hydroxide) were used to develop a gradient elution. The solvent gradient was set as follows: 3% B, 5 min; 3–8% B, 0.1 min; 8–18% B, 11.9 min; 18–32% B, 11 min; 32–45% B, 7 min; 45–80% B, 3 min; 80% B, 5 min; 80–5%, 0.1 min; and 5% B, 6.9 min. The elutes were collected every minute in a tube, and 10 fractions were pooled. All fractions were vacuum-dried and reconstituted in 0.1% (v/v) aqueous formic acid (FA) for subsequent LC–MS/MS analysis.
The LC–MS/MS analysis was performed by Novogene Bioinformatics Technology Co. Ltd. The fractionated peptides were analyzed with an EASY-nLCTM 1200 UHPLC system (Thermo Fisher Scientific) coupled to a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific) operating in data-dependent acquisition (DDA) mode. During data acquisition, the mass spectrometer was operated in positive polarity mode with a spray voltage of 2.3 kV and a capillary temperature of 320 °C. The full MS scan resolution was set to 60,000 (at 200 m/z) with an (automatic gain control (AGC) target value of 3 × 106 for a scan range of 350–1500 m/z. Forty precursors of with the highest abundances in the full scan were fragmented for higher-energy collision dissociation and subjected to MS/MS analysis with the following parameters: resolution, 15,000 (at m/z 200); AGC target value, 5 × 104; the maximum ion injection time, 45 ms; normalized collision energy, 32%; the intensity threshold, 2.2 × 104; the dynamic exclusion parameter, 60 s.
Proteomics data normalization and analysis
The MS raw data files obtained with an Q-Exactive HF-X mass spectrometer were searched with Proteome Discoverer 2.2 search engine (PD 2.2; Thermo Fisher Scientific) against the UniProt database (homo_sapiens_uniprot_2019.01.18.fasta, containing 169,425 sequences). To reduce the probability of false peptide identification, only peptides identified at the 99% confidence interval through a Proteome Discoverer probability analysis were counted. For protein quantitation, each confident protein identification contained at least one unique peptide, and the peptide false discovery rate (FDR) was no more than 1.0%. The protein quantitation results were statistically analyzed by Mann–Whitney Tests. Only a protein with a fold change ≥ 1.5 or ≤ 0.67 and a P < 0.05 was considered a significantly differentially expressed proteins (DEP).
Functional classification of DEPs was performed using a Gene Ontology (GO) enrichment analysis (http://www.geneontology.org/). Pathway enrichment analysis of DEPs was carried out using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/). The significance of a KEGG enrichment pathway with a Bonferroni corrected P < 0.05 (q value) was determined using a hypergeometric test.
Cell viability assay
Cell viability was determined using cell counting kit-8 reagent (CCK-8, Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocol. Briefly, A549 cells were seeded in 96-well culture plates at a density of 1 × 104 cells per well and cultured for 24 h at 37 °C. The cells were then infected with SIV at an MOI of 0.01, 0.1, or 1 or treated with 10 μM erastin (Selleck Chemicals, Houston, TX, USA) as a positive control. Cells were treated with or without the ferroptosis inhibitor Fer-1 (1 μM, Selleck Chemicals) 1 h before SIV infection to determine the role of ferroptosis in SIV-infected cells. After culture for 24 h, 10 μL of the CCK-8 reaction mixture were added to each well and incubated at 37 °C for 4 h. The absorbance of each well was measured at 450 nm with a microplate reader (Thermo Fisher Scientific), and the corresponding optical density ratio was expressed as cell viability.
For the analysis of cell morphology, A549 cells were seeded in 6-well plates and infected with SIV at an MOI of 1 or treated with 10 μM erastin for 24 h. Fer-1 (1 μM) was added 1 h before SIV infection to inhibit cell ferroptosis. Afterward, cells were fixed with 4% paraformaldehyde for 20 min at RT and washed with PBS. Images were captured using a Carl Zeiss (Oberkochen, Germany) converted fluorescence microscope (20 × objective).
Measurement of cellular iron level
The intracellular Fe2+ levels were detected using the iron assay kit (Abcam, Cambridge, UK). Briefly, after treatment of the different groups, cells were washed with cold PBS and homogenized in iron assay buffer for 30 min at 37 °C. Sample was then added with iron probe and incubated at 37 °C for 60 min. The absorbance was read at 593 nm. The BCA protein determination method was used for total protein quantification, and the cellular iron concentration is presented as μM/mg protein. The intracellular Fe2+ levels were also detected using FerroOrange probes (Dojindo, Japan) following the manufacturer’s instructions, and Fe2+ image of the living cells was performed using a fluorescence microscope.
Changes in intracellular ROS levels were using DCFDA/H2DCFDA in cellular ROS assay kits (Abcam). Briefly, A549 cells were cultured in 6-well plates at a density of 2 × 105 cells per well and allowed to attach overnight. After treatment, cells in groups were labeled with 20 µM DCFDA and incubated for 30 min at 37 °C. Stained cells were washed with PBS and transferred to FACS tubes. The fluorescence of each probe was detected using a CytoFLEX flow cytometer (Beckman Coulter, Suzhou, China) and analyzed by FlowJo software.
Lipid peroxidation assay
Lipid peroxidation was determined by measuring the malondialdehyde (MDA) level with a lipid peroxidation MDA assay kit (Beyotime Biotechnology). Briefly, cells were homogenized and the supernatant was mixed with thiobarbituric acid (TBA)-glacial acetic acid reagent. Then, the TBA-MDA mixture was heated at 100 °C for 1 h. The absorbance was measured at 532 nm with a microplate reader and the MDA concentration was calculated as μM/mg protein. Lipid peroxidation was also confirmed by fluorescence observations using Liperfluo (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. After an incubation with the indicated treatments, cells were stained with 5 μmol/L Liperfluo and Hoechst nuclear stain and then visualized under a florescence microscope (Carl Zeiss, Oberkochen, Germany).
Reductive GSH content and NADP.+/NADPH assay
The intracellular levels of reductive GSH and NADP+/NADPH were determined using a GSSG/GSH quantification kit (Dojindo) and an NADP+/NADPH assay kit (Beyotime Biotechnology) respectively, following the manufacturer's instructions.
A549 cells were infected with SIV at an MOI of 1 for the indicated times or treated with 1 μM Fer-1 1 h before SIV infection. The cells were collected and lysed in RIPA lysis buffer (Beyotime), denatured and loaded on 10% gels for SDS–polyacrylamide gel electrophoresis. The protein bands were then transferred onto 0.2-μm nitrocellulose membranes (Millipore, Billerica, MA, USA), blocked with 5% nonfat milk, and incubated overnight at 4 °C with primary antibodies against SLC7A11 (Cell Signaling Technology), SLC3A2L (Abcam), GPX4 (Santa Cruz), transferrin (TF) (Abcam), transferrin receptor (TFRC) (Abcam), NS1 protein (prepared in our lab) and β-actin (Cell Signaling Technology). Membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology) for 1 h. The antibody-labeled proteins were detected by chemiluminescence using SuperSignal West Pico PLUS Chemiluminescence Substrate (Thermo Fisher Scientific) with an Amersham Imager 600 (Cytiva Sweden AB, America). Densitometry analysis was performed using the ImageJ software.
Quantitative real-time PCR (qRT–PCR)
To explore the function of ferroptosis inhibitor treatment on SIV-induced inflammatory cytokine expression, A549 cells were treated with or without Fer-1 before SIV infection and then subjected to qRT–PCR analyses to measure the mRNA levels of IL-6, IL-8, IL-1β and TNF-α at 24 h.p.i.. Total RNA was extracted using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. cDNA synthesis was performed using a SuperScript III kit (Invitrogen) and oligo-dT primer. 50 ng of the cDNA product was used as the template for qRT–PCR in a final volume of 10 μl containing SYBR Premix Ex Taq II (TaKaRa, Dalian, China). qRT–PCR was performed on a 7500 Real Time PCR System apparatus (ABI, Madison, WI, USA). The amplification conditions consisted of 95 °C for 30 s and 40 cycles of 95 °C for 5 s, 60 °C for 30 s, and 95 °C for 15 s. The changes in the levels of target mRNAs are presented as fold changes and were calculated using the comparative Ct method as previously described. The following primer sequences were used for amplification:
IL-6 (F:AAGCCAGAGCTGTGCAGATGAGTA, R:TGTCCTGCAGCCACTGGTTC), IL-8 (F:TTTCAGAGACAGCAGAGCACA, R:CACACAGAGCTGCAGAAATCAG), IL-1β (F:GCTGATGGCCCTAAACAGATGA, R:TCCATGGCCACAACAACTGAC), TNF-α (F:CTCAGCAAGGACAGCAGAGG, R:ATGTGGCGTCTGAGGGTTGTT), GAPDH (F:GCACCGTCAAGGCTGAGAAC, R:TGGTGAAGACGCCAGTGGA).
Titration of viruses
A549 cells grown in 96-well plates were treated with Fer-1 and then infected with SIV at an MOI of 1. The cells were then incubated for 24 h. The supernatant of infected culture was harvested, serially diluted and titrated in MDCK cells. Virus titers were calculated by the Reed-Muench method and were expressed as TCID50 per milliliter of supernatant.
GraphPad Prism 6 software (GraphPad Software Inc., San Diego, CA, USA) was used for the data analyses and graph creation. All the data are presented as the means ± standard deviations (SD). The significance was determined with either unpaired two-tailed independent Student’s t test for comparisons between two groups or one-way ANOVA. A p < 0.05 was considered to be statistically significant.