Cell lines
All cell culture products were purchased from Gibco (Life Technologies, Grand Island, NY, USA). SJPL cell line [36] and MARC-145 cell line [37] are known to support PRRSV replication [38, 39]. SJPL cells were provided by R.G. Webster (St. Jude Children’s Hospital, Memphis, TN, USA) and were historically defined as porcine lung cells [36] but were later found to come from monkey [40]. SJPL cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 1 % (v/v) MEM non-essential amino acid (NEAA) solution, 100 μg/mL streptomycin, 100 U/mL penicillin, 50 mg/L gentamicin and 2.5 mg/L amphotericin B. MARC-145 cell line is a subclone of African green monkey kidney cell line MA104 [38]. MARC-145 cells were grown in minimum essential medium (MEM) supplemented with 10 % (v/v) FBS, 2 mM L-glutamine, 10 mM HEPES, 10 μg/mL streptomycin, 10 U/mL penicillin and 2.5 mg/L amphotericin B. Cells were cultured and infected at 37 °C in a 5 % CO2 atmosphere.
Bacterial and viral strains
Actinobacillus pleuropneumoniae non-hemolytic and non-cytotoxic MBHPP147 was kindly provided by Ruud P.A.M. Segers (MSD Animal Health, Boxmeer, The Netherlands). This strain is a mutant of the serotype 1 reference strain S4074 producing non-active ApxI and ApxII toxins (AppΔapxICΔapxIIC) [41]. Bacteria was grown at 37°C in brain heart infusion broth (BHI; Oxoid Ltd., Basingstoke, Hampshire, England) or in BHI agar (Oxoid Ltd.) supplemented with 5 μg/mL or 15 μg/mL of β-nicotinamide adenine dinucleotide (β-NAD; Sigma-Aldrich, St Louis, MO, USA), respectively.
The PRRSV North American reference strain IAF-Klop was used in this study. This strain is a genotype II strain [42, 43].
Bacterial culture supernatant
Actinobacillus pleuropneumoniae culture supernatant was prepared as recently described by Lévesque and collaborators [7]. Briefly, an overnight culture of AppΔapxICΔapxIIC was diluted into fresh BHI broth and grown to an OD600 of 0.6. This culture was then diluted into DMEM medium, supplemented with 10 % FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 % NEAA, to 1 × 106 CFU/mL and incubated overnight at 37 °C. Bacterial cells were centrifuged at 4,000 g for 20 min and the supernatant was harvested and passed through a 0.22 μm filter (Sarstedt Inc., Newton, NC, USA). Filter-sterilized supernatant was ultrafiltrated through a 3 kDa membrane (Merck Millipore Ltd., Carrigtohill, Cork, Ireland). Resulting culture supernatant ultrafiltrate and the filter-sterilized supernatant were stored at −20°C up to 6 months. Supplemented DMEM medium was also ultrafiltrated through a 3 kDa membrane and used as a control.
Protein profiling of SJPL cells
Sample preparation was performed as described by Auger and collaborators [28]. Briefly, a T25 flask (Sarstedt Inc.) of confluent SJPL cells (0.5 × 106 cells) were infected or not with IAF-Klop PRRSV strain for 4 h (multiplicity of infection (MOI): 0.5) and then treated with AppΔapxICΔapxIIC culture supernatant or left untreated for 18 h. These conditions are known to block PRRSV replication [7]. Cells were washed twice with Dulbecco's phosphate-buffered saline (DPBS; Life Technologies) and 500 μL of lysis buffer was added (20 mM MOPS (pH 7.0; Sigma-Aldrich), 2 mM EGTA (Sigma-Aldrich), 5 mM EDTA (Fisher Scientific, Fair Lawn, NJ, USA), 30 mM sodium fluoride (Sigma-Aldrich), 60 mM β-glycerophosphate (pH 7.2; Sigma-Aldrich), 20 mM sodium pyrophosphate (Sigma-Aldrich), 1 mM sodium orthovanadate (Sigma-Aldrich), 1 % (v/v) Triton X-100 (Sigma-Aldrich) and one protease inhibitor cocktail tablet Complete Mini EDTA-free (Roche Diagnostics GmbH, Mannheim, Germany), pH 7.2). Cells were removed using a cell scraper (Nalge Nunc International, Rochester, NY, USA) and transferred into a microcentrifuge tube on ice. Sonication was performed at 180 Joules using an ultrasonic processor (Cole-Parmer, Vernin Hills, IL, USA) in order to lyse the cells. Samples were then ultracentrifuged at 90,000 g for 30 min in a Sorvall RC M100 ultracentrifuge (Beckman Coulter, Mississauga, ON, Canada). Protein concentration was determined on the supernatant using Pierce BCA protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) and was adjusted to 2 mg/mL. Samples were then analyzed using Kinex KAM-850 antibody microarray (Kinexus Bioinformatics Corporation, Vancouver, BC, Canada). Kinex KAM-850 antibody microarray includes 854 antibodies which target different cell signaling pathways and included 517 pan-specific antibodies and 337 phosphosite-specific antibodies. These include 466 antibodies against protein kinase, 44 antibodies against protein phosphatase, 46 antibodies against stress response proteins, 75 antibodies against protein implicated in transcription and 223 others. Data were expressed in a chemiluminescence signal ratio versus the untreated cells. The percentage change from control (% CFC) was calculated for each protein by dividing the treated condition Z-ratio by the control Z-scores X 100 as described previously [44].
Cell cycle analysis
Cell cycle analysis was done using a modified protocol from Sazer and Sherwood [45]. Briefly, 0.5 × 106 SJPL cells were cultured overnight in T75 flask (Sarstedt Inc.) in DMEM. Cells were treated for 18 h at 37 °C in a 5 % CO2 atmosphere with either DMEM, the ≤ 3 kDa DMEM ultrafiltrate, AppΔapxICΔapxIIC undiluted culture supernatant, the ≤ 3 kDa culture supernatant ultrafiltrate, 0.4 μM nocodazole (Sigma-Aldrich), 7.4 μM aphidicolin (Sigma-Aldrich), 100 μM 3,3′-diindolylmethane (DIM; R&D Systems Inc., Minneapolis, MN, USA) or 50 μM SBE-13 hydrochloride (SBE-13; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Cells were washed twice with DPBS and then trypsinized at 37 °C for 10 min. Trypsin (Life Technologies) was inhibited using complete medium and cells were centrifuged at 380 g for 15 min at 4 °C. Cells were washed one more time with DPBS and then fixed and permeabilized using 70 % (v/v) cold ethanol for 2 h at room temperature followed by an overnight incubation at 4 °C. Cells were washed twice with DPBS then treated for 2 h at 37 °C with 2 mg of ribonuclease-A (RNase-A; Sigma-Aldrich) in 900 μL of DPBS and cells were finally stained with 100 μL of propidium iodide (PI) 0.2 mg/mL (Sigma-Aldrich). Stained cells were analyzed on BD FACS Calibur (Becton Dickinson, Mississauga, ON, Canada), measuring fluorescence emission at 585 nm after excitation at 488 nm. For each analysis, 10,000 events were evaluated and DNA content was determined by ModFit LT v3.0 (Verity software house Inc., Topsham, ME, USA), which provided histograms to evaluate cell cycle distribution.
Cell counting
To compare cell proliferation and cell viability during treatments, we used a Countess automated cell counter (Life Technologies). After the trypsinization step in the cell cycle analysis protocol, cells were washed twice in DPBS and centrifuged at 380 g for 15 min at 4 °C. Cells were resuspended into 1 mL DPBS and 100 μL of this cell suspension was added to 100 μL of trypan blue (Life Technologies) and 10 μL of this dilution was added to a Countess cell counting chamber slide (Life Technologies) to perform cells count and viability measurement. Data were automatically expressed as number of viable cells per mL.
Immunofluorescence assay
To detect PRRSV infected cells, we used a modified immunofluorescence assay (IF) protocol from Provost and collaborators which detects PRRSV antigens [39]. Briefly, confluent cells (1 × 104 cells per well in 96-well plates (Corning Inc., Corning, NY, USA)) were infected or not with PRRSV (MOI: 0.5) for 4 h and treated for 44 h with either DMEM, the ≤ 3 kDa DMEM ultrafiltrate, AppΔapxICΔapxIIC undiluted culture supernatant, the ≤ 3 kDa culture supernatant ultrafiltrate, 100 μM DIM, 0.25 % (v/v) ethanol (DIM solvent), 50 μM SBE-13 or 0.5 % (v/v) H2O (SBE-13 solvent). Following infection and/or treatment, cells were washed and then fixed for 15 min at room temperature with a 50 % (v/v) methanol (Fisher Scientific) and 50 % (v/v) acetone (J.T. Baker Inc., Phillipsburg, NJ, USA) solution. Cells were washed three times using phosphate-buffered saline without KCl (PBS): 0.1 M NaCl (Sigma-Aldrich), 4 mM Na2HPO4 (Fisher Scientific), 1.5 mM KH2PO4 (Biopharm Inc., Laval, QC, Canada) and then incubated for 90 min at 37 °C with a rabbit monospecific antisera (anti-PRRSV N protein) [43], diluted 1/150 in PBS. Cells were washed three times with PBS and incubated at 37 °C for 60 min with an anti-rabbit antiserum FITC-conjugated (Life Technologies) diluted 1/75 in PBS. Finally, cells were washed three other times with PBS and visualized using a Leica DMI 4000 inverted widefield fluorescence microscope (Leica Microsystems Inc., Richmond Hill, Canada). Pictures were acquired using DFC 490 digital camera (Leica Microsystems Inc.) and images were analyzed using Leica Application Suite Software, version 2.4.0 (Leica Microsystems Inc.).
Sample preparation for biochemical analysis
In order to eliminate salts and remove inorganic contaminants, a salting-out liquid-liquid extraction was performed. Briefly, 2 mL of ultrafiltrate samples (≤3 kDa) were mixed vigorously for 1 min with 2 g of NaCl (Sigma-Aldrich) and 2 mL of acetonitrile (Fisher Scientific), organic layer was kept after decantation for analysis.
Thin-layer chromatography
Thin-layer chromatography (TLC) plates were used to analyze samples and preparative TLC plates were used to isolate molecules of interest. For samples analysis, 40 μL of the ≤ 3 kDa ultrafiltrate were applied on a 5 cm × 10 cm (length × width) silica gel plate (Sigma-Aldrich). For preparative TLC plates, 1 mL of the ≤ 3 kDa ultrafiltrates were applied on a 20 cm × 20 cm (length × width) silica gel plate (Sigma-Aldrich). Samples were separated with a mobile phase composed of 75 % (v/v) chloroform (BDH Inc., Toronto, Ontario, Canada) and 25 % (v/v) methanol (Fisher Scientific) in a saturated-closed glass chamber. Migration was performed at ambient temperature (21 °C ± 3 °C). After migration, plates were air-dried for 5 min and then observed under a ultra-violet A (UV-A) light (Ultra-Lum Inc., Claremont, CA). Areas which correspond to spots of interest were scraped from the silica gel plate and transferred into a microcentrifuge tube. The extracted silica was weighed and molecules were extracted with acetonitrile at a ratio of 1:3 (w/v) for 2 h at room temperature. Samples were then centrifuged at 18,000 g for 5 min and the supernatant was kept for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.
Instrumentation and bioanalytical methods
The LC-MS/MS system included a Thermo Surveyor autosampler, a Thermo Surveyor MS pump and a Thermo LCQ Advantage Ion Trap Mass Spectrometer (Thermo Scientific, San Jose, CA, USA). Data was acquired and analyzed with Xcalibur 1.4 (Thermo Scientific). Samples were analyzed by LC-MS/MS in full scan positive ion mode. Briefly, 2 μL of sample was injected and the separation was achieved with a gradient mobile phase along with a microbore column Thermo Biobasic C8 (100 × 1 mm) (Thermo Scientific), with a particle size of 5 μm. The initial mobile phase condition consisted of acetonitrile and water (both fortified with 0.4 % (v/v) of formic acid) at a ratio of 5:95. From 0 to 1 min, the ratio was maintained at 5:95. From 1 to 21 min, a linear gradient was applied up to a ratio of 90:10 and maintained for 5 min. The mobile phase composition ratio was then reverted to the initial conditions and the column was allowed to re-equilibrate for 14 min for a total run time of 40 min. The flow rate was fixed at 75 μL/min. Mass spectrometer was coupled with the LC system using a pneumatically assisted electrospray ion source (ESI). Sheath nitrogen gas was set to 10 units and ESI electrode was set to 4000 V in positive mode. Capillary temperature was set at 300 °C and capillary voltage to 34 V. Instrument was operating in full scan MS mode (m/z 200–1000) and MS/MS data was collected on the most abundant peaks in a data dependent acquisition (DDA) mode using a peak intensity threshold of 1 × 105 counts per second (cps). The ≤ 3 kDa ultrafiltrate of DMEM was used as a negative control and spectra were compared with the ≤ 3 kDa ultrafiltrate of the A. pleuropneumoniae culture supernatant or the TLC extracted molecules.
Statistical analyses
For cell cycle data analyses, an arcsine square root ASIN(SQRT(x)) transformation was performed before statistical analysis. When n was equal or above eight, a d’Agostino and Pearson omnibus normality test was then utilized, followed by a one-way analysis of variance (ANOVA) with Dunnett post-test or a paired t-test to determine if significant differences exists between the different cell cycle phases or the cell-counting results of untreated and treated cells. All statistical analyses were performed using GraphPad PRISM v5 (GraphPad Software Inc., San Diego, CA, USA). Significant differences were considered when P ≤ 0.05.