Cell culture and identification

Primary explant-derived HTMCs (iCell Bioscience Inc., China) were grown in DMEM/F12 with 20% serum and kept at 37 °C in a 5% CO2 environment. Only cells in the 3rd to 5th passages were used. To identify TMCs, collagen type IV, laminin and fibronectin antigens were detected by immunocytochemistry.

Sample collection and protein preparation

TMCs were pretreated with SOD (0, 1, 5, or 10 U/mL) for 30 min and then exposed for 24 h to a range of H2O2 concentrations (0, 50, 100, 150, and 200 μM). The concentrations of H2O2 and SOD in the group with the most obvious protective effect of SOD were screened through a Cell Counting Kit-8 (CCK-8) assay. The cells were divided into three groups: the control group (without any interference, group C), the H2O2-treated group (with exposure to H2O2 only, group H), and the SOD pretreatment group (with exposure to SOD before H2O2 addition, group S). The cells were collected and stored at − 80 °C after homogenization and centrifugation. Then, a mammalian tissue total protein extraction kit (AP0601-50) was used to extract proteins from the different groups. The protein concentrations were estimated using a Bradford assay kit. Next, 20 µg of protein from each sample was mixed with 5× loading buffer at a ratio of 5:1 (v/v). The supernatant was collected after 5 min of boiling in a water bath and 10 min of centrifugation at 14,000×g. The proteins were separated on a 10% SDS-PAGE gel (constant current of 14 mA, 90 min). Coomassie Blue R-250 staining was used to visualize the protein bands. After quantification, two hundred micrograms of protein from each sample was incorporated into 30 μL SDT buffer (4% SDS, 100 mM DTT, 150 mM Tris–HCl pH 8.0) and incubated at 37 °C for 1 h. DTT and other low-molecular-weight components were removed using UA buffer (8 M urea, 150 mM Tris–HCl pH 8.5) by repeated ultrafiltration (Sartorius, 10 kD). Then, 100 μL iodoacetamide (100 mM IAA in UA buffer) was added to block reduced cysteine residues, and the samples were incubated for 1 h in darkness. After that, we added 100 μL of NH4HCO3 (50 mM) to dilute the UA, mixed the samples and centrifuged them under the same conditions. The supernatant was removed as before; this step was repeated three times. Finally, we replaced the collection tube with a new collection tube, added trypsin at a ratio of 50:1 (protein:trypsin) to the digested proteins and incubated the samples at 37 °C for 16 h. After digestion, the peptides were vacuum dried, dissolved in 0.1% trifluoroacetic acid (TFA), desalted on C18 cartridges (Empore SPE Cartridges C18, standard density), dried by vacuum centrifugation and reconstituted in formic acid (FA).

LC–MS/MS analysis

The reconstituted peptides were analyzed with a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a nano high-performance liquid chromatography (UltiMate 3000 LC Dionex; Thermo Fisher Scientific) system. After protease hydrolysis, 800 ng of proteins from different samples were pressure-loaded onto a C18-reversed-phase column (3 μm-C18 resin, 75 μm × 15 cm) and separated on an analytical column (5 μm C18 resin, 150 μm × 2 cm) using mobile phases A: 0.5% formic acid [FA]/H2O and B: 0.5% FA/ACN at a flow rate of 300 nL/min. The chromatographic separation gradient is shown in Table 1. Spectra were acquired in data-dependent mode. The 10 most intense ions selected for MS scanning (300–1800 m/z, 60,000 resolution at m/z 400, accumulation of 1 × 106 ions for a maximum of 500 ms, 1 microscan). The isolation window was 1.3 m/z, and the MS/MS spectra were accumulated for 150 ms using an Orbitrap. MS/MS spectra were measured at a resolution of 15,000 at m/z 400. Dynamic precursor exclusion was allowed for 120 s after each MS/MS spectrum measurement and was set to 17,500 at m/z 200. The normalized collision energy was 30 eV, and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at the maximum fill time, was defined as 0.1%. The instrument was run with peptide recognition mode enabled.

Table 1 Chromatographic separation gradient for LC–MS/MS analysis

Data analysis and bioinformatics analysis

MaxQuant (1.6.17) was used to search the reviewed FASTA database in UniProt with Homo sapiens as the organism. The following options were used to identify the proteins: peptide mass tolerance = ± 15 ppm, MS/MS tolerance = 0.02 Da, enzyme = trypsin, missed cleavage = 2, fixed modification: carbamidomethyl (C), variable modification: oxidation (M), database pattern = decoy. The false discovery rate (FDR) for peptides and proteins was set to 0.01. The protein expression data are presented in a heatmap. The DEPs between groups were defined as significantly upregulated or downregulated on the basis of a fold change (FC) ≥ 1.5 and P value < 0.05 (upregulated) or a FC ≤ 0.667 and P value < 0.05 (downregulated) (experimental group/control group). We used Metascape, a web-based resource (http://metascape.org), to conduct Gene Ontology (GO) analysis and used the Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology-Based Annotation System (KOBAS) online analysis tool (http://kobas.cbi.pku.edu.cn/) to perform KEGG pathway analyses. Database enrichment analysis was performed using the UniProtKB database (Release 2016 10). GO enrichment included three ontologies (biological process (BP), molecular function (MF), and cellular component (CC)). In addition, we performed protein–protein interaction (PPI) analysis using STRING software (http://string-db.org/) and then imported the results into Cytoscape software (http://www.cytoscape.org/, version 3.8.2) to further analyze functional PPI networks. EVenn (http://www.ehbio.com/test/Venn/#/) was used to create Venn diagrams.

Cell transfection with siRNAs

SiRNA against intracellular adhesion molecule-1 (ICAM-1) (siICAM-1) was provided by GenePharma (Shanghai, China) and transfected into TMCs using Lipofectamine 2000 reagent after induction with H2O2 for 24 h. The sense strand of siICAM-1 used for gene knockdown was as follows: 5ʹ-GCCAACCAAUGUGCU AUUCAAdTdT-3ʹ. The scrambled siRNA sequence was UUCUCCGAACGUGUCACGUdTdT. Before transfection, the TMCs were cultured in 6‐well plates with complete medium for 24 h. siICAM-1 was transfected with Lipofectamine 2000 (Thermo) in serum‐free DMEM for 6 h, and then, the mixture was replaced with complete medium. Western blotting was used to verify protein knockdown 24 h post-transfection.

Western blotting

TMCs were lysed with lysis buffer (20 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L EGTA, 1 mmol/L EDTA, 10% glycerol, 1 mmol/L MgCl2, 1% Triton X‐100). The extracts were centrifuged at 12,000 rpm for 20 min at 4 °C, and the supernatants were collected. The protein extracts were separated on 10% polyacrylamide‐SDS gels, transferred to PVDF membranes and then blocked with 5% skimmed milk powder for 1 h. After incubation with primary antibodies (anti-ICAM, Abcam) overnight, the membranes were washed three times and incubated with fluorescent secondary antibodies at room temperature for 2 h. The fluorescent signals were captured using an infrared imager (Millipore, USA).

Cell viability assay

TMC viability was examined using a CCK-8 (Dojindo Molecular Technologies, Gaithersburg, MD, USA). TMCs were seeded in 96-well plates and incubated at 37 °C for 24 h. After cell transfection and/or stimulation with H2O2, the culture medium was replaced with TMC medium (TMCM) containing 10% CCK-8 solution, and the cells were incubated at 37 °C for an additional 2 h. Finally, the absorbance at 450 nm was detected by using a microplate reader (Bio-Rad, Hercules, CA, USA).

Statistical analysis

The two-tailed Student t test was used for statistical analysis. All data are expressed as the mean ± SE, and a P value < 0.05 was considered to indicate statistical significance.

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