Effect of downregulated citrate synthase on oxidative phosphorylation signaling pathway in HEI-OC1 cells

Cells and the main reagents

HEI-OC1 (House Ear Institute-Organ of Corti 1) Cells were donated by F. Kalinec of House College of Ear Sciences in the United States and kept in the Key Laboratory of Genetic Hearing Disorders of Shandong Province. shRNACs-1429 cells with pGLV-H1-GFP + Puro-Cs1429 which contain shRNA sequences (5′-GCATGACGGAGATGAACTACT-3′) targeting mouse Cs gene were the low-expression Cs cell model constructed from HEI-OC1 cells in our laboratory, shRNA-NC cells with pGLV-H1-GFP + Puro-NC as negative control [5].

Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco USA. iTRAQ Reagent form AB Sciex LLC (USA). Other reagents are from Sangon Biotech (Shanghai, China) Co., Ltd.

Cell culture and total protein extraction

Cells were cultured in high-glucose DMEM (FBS, 10%) medium at 33 °C and 10% CO2. The medium was changed every 2–3 days. Logarithmic growth phase cells were collected for lysis on ice with RIPA (PMSF, 1 mM) buffer. The collected cells were broken by ultrasound under low temperature with power of 80 W, ultrasound 1.0 s, and turned off 1.0 s, for a total of 3 min. After centrifugation at 12000 r·min− 1 and 4 °C for 20 min, the supernatants were collected as the total protein extract. The total protein concentration was determined by BCA protein assay (PC0020, Solarbio).

Proteolysis and iTRAQ labeling

Total protein extraction (100 μg) was reacted with 120 μL reducing buffer (10 mM DTT, 8 M Urea, 100 mM TEAB, pH 8.0) on 10 K ultrafiltration tube at 60 °C for 1 h, and then reacted with the alkylation reagent iodoacetamide (IAA, 50 mM) in the dark for 40 min at room temperature. The filter was washed with 100 μL TEAB (100 mM) twice. 2 μl sequencing-grade trypsin (1 μg/μL) was used to digest proteins on filter of each tube at 37 °C for 12 h. The hydrolysis products were labeled with iTRAQ reagent (ABSCIEX) at room temperature for 2 h. The reaction was terminated by 200 μL of HPLC water for each sample. The labeling peptides solutions were lyophilized and stored at − 80 °C.

Analysis and identification of reverse phase chromatography-mass spectrometry

The labeled polypeptide mixture was separated by reversed-phase chromatography. The mobile phase A was H2O-FA(99.9:0.1, v/v) and the mobile phase B was ACN-H2O-FA (80:19.9:0.1, v/v/v). The obtained samples were sampled on a C18 precolumn (PepMap C18, 100 μm × 2 cm, 5 μm) at a flow rate of 300 nL/min. The peptide segments were then gradient eluted with an analytical column (PepMap C18,, 75 μm × 50 cm, 2 μm) and detected by mass spectrometry. Cellular protein extractions and mass spectrometry analysis were performed in triplicate.

Protein identification and quantification

The data were analyzed with The software Proteome DiscovererTM 2.2 (Thermo Company of the United States) on the basis of uniprot-Proteome_UP000000589-mus Musculus (Mouse) (Strain C57BL6J) database. The false-positive rate of peptides identification was controled under 1%. The parameter for searching library was set to: 1) Sample type: iTRAQ 8 plex (Peptide Labeled); 2) Cysteine alkylation: iodoacetamide; 3) Digestion: trypsin; 4) Trusted proteins: screening according to Score Sequest HT > 0 and unique peptide ≥1, and the blank value was removed.

Bioinformatics analysis

Based on the screened trusted proteins, the FC value and p-value of difference significance of the comparison group were calculated. FC > 1.1 or FC < 10/11 and p-value < 0.05 were used as the standard to screen the trusted proteins with significant difference. Functional classification and GO enrichment analysis and KEGG pathway analysis were constituted by integration analysis cloud platform based on OmicsBean omics data. GO functional annotation includes three levels of analysis(http://www.geneontology.org): Biological Process, Cellular Component and Molecular Function. Target pathway analyses were identified with KEGG (https://www.genome.jp/kegg/). The functional PPI network was explored in STRING (http://string-db.org/) and pictured by Cytoscape (v3.7.1) software.

Immunoblotting

The processes of the extraction of total proteins from cells and the concentration determination of total proteins were conducted as above. Equal amounts of proteins in each sample were loaded onto 10% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel following with electrophoresis. Proteins were then transferred to polyvinylidene fluoride (PVDF) membrane (microporous). The membranes were incubated at 4 °C overnight with antibodies against Cs (ab96600, Abcam), Ndufb5 (23855–1-AP, Proteintech), Ndufv1(11238–1-AP, Proteintech) and Uqcrb(10756–1-AP, Proteintech) or β-actin (ab8227, Abcam) at a dilution of 1: 1000. Then the membranes were washed three times with TBST, followed with incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ab97051 1:5000, Abcam) at room temperature for 2 h. The bands of specific proteins were visualized by ECL kit (NOVLAND, Shanghai) and photographed in the chemiluminescence instrument (Clinx Science Instruments Shanghai, China). The intensity of every protein band was quantified by Image J software with that of β-actin as control.

Intracellular ATP analysis

ATP levels of cells were measured using the ATP Assay Kit (Beyotime Biotechnology, Shanghai, China). Logarithmic growth phase cells were seeded into 6-well plates. 200 μL of lysis buffer for every well was added to lyse the cells when cells grew to 80%. The supernatant was collected and mixed with 100 μL of ATP test working solution in a black 96-well plate. Luminescence was measured by luminometer (Infinite M200 Pro, Tecan, Switzerland). The standard curves were generated with ATP standard solution (concentrations: 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 μM) for each experiment. The ATP concentration was determined according the standard curve and standardized by protein concentration (BCA Protein Assay Kit, Beyotime Biotechnology, Shanghai, China).

Statistical analysis

Statistical analysis was carried out by GraphPad Prism 9 software (GraphPad Software, San Diego, CA, USA). All the bars in the graph represent the means ± SEM from at least three independent experiments. For determining the significance of differences between two groups, Student’s t-test was applied. P-value < 0.05 was considered as statistically significant difference.

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