Cell culture and stable Plac9-overexpressing clones
Human bronchial epithelial cell line 16HBE was purchased from Shanghai Zhongqiaoxinzhou Biotech (Catalog Number: ZQ0001). 16HBE cells were cultured in Dulbecco’s modified Eagle Medium (DMEM, Hyclone, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, ScienCell, San Diego, CA, USA) in 5% CO2 at 37 °C. Stable Plac9-overexpressing cell line (16HBE-GFP-Plac9) and the corresponding stable control (16HBE-GFP) were custom-made by Genechem Co., Ltd, Shanghai, China.
Protein preparation and digestion
The cells were randomly pooled into three groups. The three replicates were treated for iTRAQ analysis to identify differentially expressed proteins as previously described with minor revision [49, 50] . Each sample was sonicated for 15 min in 500 μL SDT lysis buffer (4% sodium dodecyl sulfate (SDS), 100mM Tris-HCl, pH 7.6). Samples were then incubated in water for 15 min at 95 ℃ and centrifuged at 14,000 × g for 15 min. The supernatant was collected, and protein concentration was determined with bicinchoninic acid (BCA) protein quantity method. Protein samples were then stored at -80 ℃ before further analyses. 30 μL of protein mixtures was mixed with 1 M dithiothreitol (DTT) at a final concentration of 100 mM, and then incubated in 95 ℃ water bath for 5 min. After cooling to room temperature, the sample was mixed with 200 μL UA buffer (8 M urea and 150 mM Tris - HCl, pH 8.5), loaded onto an ultrafiltration filter (30-kDa cutoff, Sartorius, Germany), centrifuged at 12,500 × g for 25 min, and the filter was washed with UA buffer twice. Subsequently, 100 μL of iodoacetamide solution (100 mM iodoacetamide in UA buffer) was added to the filter, vortexed for 1 min at 600 × rpm, incubated for 30 min at room temperature in the dark, and centrifuged at 12,500 × g for 25 min. Filtrate was discarded. The filter were washed twice with 100 μL UA buffer (12,500 × g, 15 min). Next, 100 μL dissolution buffer (Applied Biosystems, Foster City, CA, USA) was added and centrifuged at 12,500 × g for 15 min. This step was repeated twice. Then 40 μL trypsin (Promega, Madison, WI, USA) buffer (5 μg trypsin in 40 μL dissolution buffer) was added to the filter and the filter was softly vortexed for 1 min at 600 × rpm. The filter was incubated at 37 °C for 16 -18 h. The filter unit was transferred to a new tube and centrifuged at 12,500 × g for 15 min. The digested peptides were collected with 20 μL dissolution buffer and the peptide concentration was measured on a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) at 280 nm.
iTRAQ labeling and analysis
A total of 100 μg peptide mixture was labeled with iTRAQ reagents according to manufacturer instructions (Applied Biosystems, Foster City, CA, USA). Triplicate 16HBE-GFP-Plac9 samples were labeled with reagent 114, 115, and 116, respectively. Triplicate 16HBE-GFP samples were labeled with reagent 117, 118, and 121, respectively. The labeled samples were mixed and fractioned on Agilent 1260 infinity II HPLC system (Agilent Technologies, Palo Alto, CA, USA). Buffer A consisted of 10 mM HCOONH4, 5% (v/v) acetonitrile, pH 10.0; Buffer B consisted of 10 mM HCOONH4, 85% (v/v) acetonitrile, pH 10.0. The column was equilibrated with buffer A, and the samples were separated in 0% (v/v) buffer B for 25 min, 0 - 7% (v/v) buffer B for 5 min, 7 - 40% (v/v) buffer B for 35 min, 40 - 100% (v/v) buffer B for 5 min, and 100% (v/v) buffer B for 15 min at a flow rate of 1 mL/min. Approximate 36 samples were collected, lyophilized, dissolved with 0.1% formic acid (FA), and pooled into 10 fractions for further mass spectrometry analysis.
Each fraction was separated on Easy nLC system (Thermo Fisher Scientific, San Jose, CA, USA). Buffer C consisted of 0.1% (v/v) formic acid in MilliQ water; buffer D was buffer C with 80% (v/v) acetonitrile. Then samples were loaded via an autosampler onto the analytical column AcclaimTM PepMapTM RSLC 50 μm × 15 cm, nano viper (Thermo Fisher Scientific, San Jose, CA, USA) and separated at 300 nL/min.
Peptide analysis was performed on a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) in positive ion mode for 120 min, with a selected mass range of 350 - 1,800 mass/charge (m/z). For the survey scan, a first-order mass spectrum resolving power was set to 70,000, an automatic gain control (AGC) target value was 3E6, a first-order maximum ion injection (IT) time was 50 ms. The m/z ratios of polypeptide and polypeptide fragments were obtained according to the following methods: Ten-fragments mass spectrometry spectra (MS2) scan were collected after each full scan. The MS2 Activation Type was higher-energy collisional dissociation (HCD). The isolation window was 2 m/z. The second-order MS resolution was 17,500 with 1 microscan. The second-order Maximum IT time was 45 ms. The normalized collision energy was 30eV.
Bioinformatics and multivariate analyses
The raw data of the mass spectrometry (MS) analysis were derived from RAW files. Database searches and quantitative analyses were performed by using Mascot 2.6 and Proteome Discoverer 2.1 (Thermo Fisher Scientific, San Jose, CA, USA). For protein-abundance ratios measured using iTRAQ, a 1.2-fold change between two sample groups was set as the threshold and a relative quantification p-value below 0.05 were regarded as being differentially expressed. The database (Uniprot_HomoSapiens_161584_20180123) was downloaded from UniProt website (http://www.uniprot.org) on 2018-01-23. The localized sequence alignment software NCBI Basic Local Alignment Search Tool (BLAST 2.2.28+-win32.exe; http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to perform sequence alignment between the identified proteins and protein sequences in the UniProt database. The mapping function of Blast2GO Command Line (www.geneontology.org; version go_201504.obo) was used to extract GO function entries correlated with the aligned sequences for all differentially expressed proteins. The Kyoto Encyclopedia of Genes and Genomes (KEGG) Automatic Annotation Server (KAAS; http://www.genome.jp/tools/kaas/) was used to align target proteins in the KEGG GENES database . The heatmap was visualized by the online ClustVis tool (https://biit.cs.ut.ee/clustvis/) . The network of protein-protein interactions was mapped using the online STRING 10.5 tool (http://string-db.org).
Reverse transcription and quantitative real-time PCR (RT-qPCR)
The mRNA levels were determined with quantitative real-time PCR analysis by using a QuantStudio® 5 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Briefly, total RNA was extracted from cells by using an RNA extraction kit (Bioteke, Beijing, China) and cDNA was synthesized by using a cDNA synthesis kit (Thermo Fisher Scientific, San Jose, CA, USA). Then 5 ng cDNA was amplified with indicated primers (Table 1) in SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan). The PCR cycle was: 95 °C for 30 sec, 45 cycles of 95 °C for 5 sec, and 60 °C for 30 sec. The PCR amplification was followed by melting curve analysis by using the defaulted program of the QuantStudio® 5 Real-Time PCR machine (Thermo Fisher Scientific, San Jose, CA, USA). The mRNA expression levels of indicated genes were calculated relative to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, internal control) using the 2-ΔΔCt method.
MTT assay and colony formation
Cells were treated with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, St Louis, MO, USA) for cell proliferation assay as described [25, 53]. Briefly, the cells were cultured by seeding 1,000 cells/well into a 96-well tissue culture plate. Then 20 µL MTT reagents were added to each well for 4 h at 37 ˚C. After adding 200 µL dimethyl sulfoxide (DMSO) for each well, absorbance was quantified at 490 nm 0, 24, 48, 96 and 120 h later, respectively. In colony formation assay, approximately 800 cells were plated and cultured in 6-well plates, then fixed with ethanol and stained with 0.5% crystal violet 2 weeks later. The colonies were photographed and quantified.
Flow cytometer analysis
This was performed similarly as previously described [25, 53]. Briefly, approximately 106 16HBE cells were pelleted and fixed with 70% ethanol for 1 hour at 4 ˚C. After resuspending with staining buffer (2 mg/mL propidium iodide (PI): 10 mg/mL RNase: 1 × phosphate buffered saline (PBS) = 25: 10: 1000), the cell cycle distribution was assessed using a BD FACS Calibur Flow Cytometer (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. For the apoptosis assay, approximately 106 16HBE cells were harvested, resuspended and labeled with fluorescein isothiocyanate (FITC)-Annexin V and PI in the dark at room temperature for 15 min. Apoptotic cells were analyzed using a BD FACS Calibur Flow Cytometer according to manufacturer’s instructions.
Wound-healing and invasion assays
Wound-healing and invasion assays were performed as reported . 4 × 105 16HBE-GFP-Plac9 or 16HBE-GFP clones per well were seeded in a 12-well plate with complete medium (DMEM containing 10% FBS). After growing to 90% confluence, a wound was created by scraping the cell layer with a sterile pipette tip. After wounding, floating cells were removed by replacing the culture medium. Digital images of the wounds were photographed at 0, 12 and 24 h, respectively. The healing rate (100%) at different time point was calculated by comparing the recovered wound width with the starting wound width.
For cell invasion assay, 2 × 105 16HBE-GFP-Plac9 or 16HBE-GFP cells per well in 100 µL medium (DMEM supplemented with 1% FBS) were seeded in the top chambers of 24-well transwell plates (8.0 µm, pore size; Corning Costar, Corning, NY, USA), and DMEM supplemented with 10% FBS was added to the bottom chamber. Cell were maintained at 37 °C for 48 h. Then the cells on the top side of the filter were wiped out with a cotton swab, while those on the bottom side (migrated) were fixed with 4% polyoxymethylene, stained with a Giemsa staining solution (Yeasen, Shanghai, China) to visualize the cells and photographed. Five randomly selected fields per well were photographed. The numbers of cells in 5 random fields per well were counted.
Western blot analysis
Cells were collected and lysed in protein lysis buffer (Beyotime, Shanghai, China) supplemented with PMSF (Beyotime, Shanghai, China) and protease inhibitor cocktail (Bimake, Houston, TX, USA). Proteins samples (20 μg per lane) were separated on SDS-10% polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose (NC) membranes. The membranes were blocked with 5% nonfat dry milk and incubated with indicated antibodies (Cell Signaling Technology, Beverly, MA, USA) to detected the corresponding proteins by following the manufacturer’s instructions. The bands were visualized by the ECLÔ reagents (Thermo Fisher Scientific, San Jose, CA, USA). b-actin was employed as an internal control.
Differences between groups were analyzed for statistical significance by using a student's t-test. GO or KEGG enrichment analyses were performed using a Fisher’s Exact Test. All experiments were repeated at least three times. Results were expressed as means ± Standard deviation (SD). p < 0.05 represents significantly different.