2.1 Cell lines and cell culture
PC-1 cells grew as islet-like colonies of cells, whereas PC-1.0 cells grew as single cells. The source and incubation conditions of the cells were described previously .
Acetonitrile (ACN) and methanol were purchased from Merck Company (Germany); glacial acetic acid, from Damao Chemical Reagent Factory in Tianjin; and bovine serum albumin (BSA), from Sigma-Aldrich Company (USA). Trypsin (bovine pancreas), formic acid, trifluoroacetic acid, urea, protease inhibitor cocktail, dithiothreitol, trichloroacetic acid, acetone, and iodoacetamide were purchased from Sigma–Aldrich (St. Louis, MO, USA). All experimental water was purified by a Milli-Q system (Millipore Corporation, USA).A Thermo SEC120 HPLC column (5μm, 120Å) was used.An Ultimate 3000 chromatograph and Thermo LTQ-Orbitrap mass spectrometer were used for detection.
2.3 Effects of serum-free conditioned medium from PC-1.0 cells on the activity of PC-1 cells
Preparation of serum-free conditioned medium: Three methods were used to prepare conditioned medium from PC-1.0 cells, which was used to treat cultured PC-1 cells for 24 hours; then, morphological changes in PC-1 cells were observed. The following methods were used: Method 1: PC-1.0 cells were washed 5 times with PBS; Method 2: PC-1.0 cells were washed 3 times with PBS and incubated 2 times with phenol-free medium (Gibco, Grand Island, NY) for 20 minutes each; And Method 3: PC-1.0 cells were incubated in 2% PBS+phenol-free medium for 20 min and in phenol-free medium 4 times for 20 min each. The supernatants of the above samples were extracted and used to prepare the culture medium.
2.4 Extraction of total protein from samples
PC-1.0 cell and PC-1 cell supernatants and RPMI 1640 medium (negative control group) were extracted as samples 4, 5 and 6 in the serum-containing medium experimental group. Each sample was spun at 12000r/min through a 0.22μm fiber filter and concentrated using a 3 kDa concentrating tube by centrifuging at 3500 ×g for 120 min. The protein concentration was measured using the BCA method.
2.5 SEC-RPLC-MS/MS analysis
Low-abundance proteins were enriched on a size exclusion chromatography (SEC) column. The 200μl sample was washed for 10 min with buffer A at a flow rate of 0.5 ml/min. After collecting the effluent components, the remaining fractions were eluted with buffer B at a flow rate of 1ml/min for 7 min, and the collected fractions were stored at -20°C for use. The effluent components collected were centralized in a rotary concentrator with a 5 kDa molecular weight cutoff membrane and centrifuged at 10 °C for 5000 r/min. Samples were collected for subsequent application.
An Ultimate 3000 chromatograph and a Thermo LTQ-Orbitrap mass spectrometer were used for detection. Peptides were loaded on an in-house-packed C18 capillary trap column (150 μm i.d. × 4 cm) and separated using a C18 separation column (75 µm i.d.×15 cm). Phase A was 98% H2O + 2% ACN with 0.1% FA, and phase B was 2% H2O + 98% ACN with 0.1% FA. The gradient was as follows: 0-6% phase B for 10 min, 6-35% phase B for 100 min, 35-80% phase B for 10 min, and 80% phase B for 10 min. The temperature of the ion transfer capillary was set at 275°Cwith a spray voltage of 2.7 kV. The scan range was set from m/z= 300–1800.There was a 20 s exclusion window. Raw spectrum data were searched using Mascot (2.3.2). For classifying the obtained protein results, the database species used in the experiment were both hamster and bovine. Mass tolerances were set at 7ppm for parent ions and 20ppm for fragments. The fixed modification was cysteine alkylation, and the variable modification was methionine oxidation. The maximum number of missing cleavage sites was 2, and the FDR was controlled at less than 1%.
2.6 Bioinformatic analysis
Because special structural characteristics of secreted proteins usually include a signal peptide, SignalP4.1 software was used to search the current hamster protein database and to construct the hamster secreted protein database (http://www.cbs.dtu.dk/services/SignalP/, probability > 0.90) . In the serum-containing medium group, RPMI 1640 medium was used as the negative control to eliminate the error caused by unlabeled samples. The results were screened from the database of hamster secretory proteins. Subsequently, the DAVID (http://david.abcc.ncifcrf.gov/)  and STING (https://string-db.org/)  bioinformatics software tools were used to analyze protein functions and possible interacting proteins. Finally, Survival analysis of patients with the different DFs was analyzed with Kaplan Meier Plotter (http://kmplot.com/analysis/index.php?p=service&cancer=pancancer_rnaseq) . The Gene Expression Profiling Interactive Analysis (GEPIA) database was used to analyze the expression of the target genes in the TCGA database (http://gepia.cancer-pku.cn/) .The expression level of YWHAG in different cancer stages was analyzed with the online analysis platform UALCAN (http://ualcan.path.uab.edu/index.html) .
2.7 YWHAG uptake assay
Western blotting was performed as described previously . Primary antibodies against YWHAG and β-actin (Abcam, USA) were used. Samples with equivalent amounts of total protein (20 μg) were loaded. Western blot signals were quantified using an Amersham Imager 600 (GE Healthcare, Little Chalfont, UK), and band signals were expressed as relative protein amounts compared to β-actin. The purified supernatant from PC-1.0 cells were added to PC-1 cells at 60%-70% confluence. The YWHAG protein level of the PC-1 cells was tested by western blot analysis after another 24 hours of culture. Human pancreatic cancer cell lines AsPC-1 and Capan-2, which have morphological and functional characteristics similar to PC-1.0 and PC-1 cells, respectively, were used to determine if the results from hamster cells coincide with human pancreatic cancer cell lines.
2.8 Statistical analysis
Statistical analyses were conducted and graphics were generated using GraphPad Prism 6.0. P < 0.05 was considered statistically significant in this study. Comparisons of quantitative data were made using Student’s t-test.