Reagents
TDCA was purchased from New Zealand Pharmaceuticals Ltd. (Palmerston North, New Zealand). KH7 (Ann Arbor, Michigan, USA) was dissolved in DMSO. ATP and BzATP (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in PBS. 2% (v/v) Oleic acid (Croda, 4-3 Hitotsubashi 2-chome, Chiyoda-ku, Tokyo, Japan) and 4% (v/v) Monoolein (Gattefosse, Saint-priest, Cedex-France) were used to prepare the TDCA oral formulation.
Preparation of Aβ1-42 oligomer
Lyophilized 0.1 mg vial of Amyloid β Protein Fragments1-42 (Aβ) (Sigma-Aldrich) were dissolved into 50 µl DMSO (Sigma-Aldrich) for 1 h at room temperature with continuous rotation. Oligomeric Aβ1-42 was prepared by diluting the dissolved Aβ into 100 µM concentration using DMEM/F12 media. The resulting solution was then incubated for 24 h at 4°C with continuous rotation.
Isolation of Primary Microglia and Culture
Primary microglia were obtained from cerebral cortices and hippocampi of male and female one~two-day-old B6 mice or adult B6, GPCR19-/-, and P2X7R-/- mice (Jackson Laboratory, USA) using a mouse brain dissociation kit (Miltenyi Biotec GmbH, Bergisch, Gladbach, Germany). The tissue was cut into fragments using a scalpel blade (Medicom, Los Angeles, CA, USA) and then homogenized using gentleMACS(TM) C tube and gentleMACSTM Octo Dissociator (Miltenyi Biotec GmbH). Single cell suspensions were obtained after passing the tissue homogenates through MACSR Smart Strainers (70 µm) (Miltenyi Biotec GmbH). The myelin was depleted using a debris removal solution supplied with the kit. Microglia cells were isolated using CD11b MicroBeads, LS Columns, and a MACS multi stand system (all obtained from Miltenyi Biotec GmbH). Isolated microglia were washed with PB buffer (0.5% FBS in PBS) and culture using DMEM-F12 media with 20% FBS and 10 ng/ml M-CSF (PeproTech, Rocky Hill, NJ) in a 95% air and 5% CO2 atmosphere at 37°C for 48 h to stain cells with antibodies or for 24 h to stimulate cells.
For inflammasome activation, primary microglial cells from one~two-day-old B6 mice were seeded on 12 mm microscope cover glasses (Deckglaser, Luda-Konlgshofen, Germany) in 24 well PDL-coated cell culture plates (Corning, Wujiang, Jiangsu, China) at 7.5 x 104 cells/ml per well for 24 h, then serum starved for 10 h and treated with Aβ (2 µM), TDCA (400 ng/ml) for 24 h, and ATP (1 mM) for the final hour.
Cell culture
BV2 cell (Murin Microglial cell line) was maintained in a 95% air and 5% CO2 atmosphere at 37°C in DMEM (Dulbecco’s modified Eagle’s medium complete media) (Invitrogen, Carlsbad, CA, USA) containing 10% FBS (Fetal Bovine Serum) (Invitrogen), and 1% penicillin and streptomycin (Invitrogen). BV2 cells were seeded in 6 well (1.5 x 105 cells/2 ml), 12 well (1 x 105 cells/ml), or 24 well (5 x 104 cells/0.5 ml) PDL-coated cell culture plates for 12 h using DMEM complete media. Cells were serum starved before treatment for 10 h using DMEM/F12 media (Thermo Fisher Scientific, Waltham, MA, USA). Cells were treated with Aβ (2 µM), TDCA (400 ng/ml), and ATP (1 mM) for 1 h in the case of GPCR-19, P2X7R interaction. For inflammasomal activation and inhibitor (KH7) assays, cells were treated with Aβ (2 µM), KH7 (4 µM), TDCA (400 ng/ml) for 24 h, and ATP (1 mM) for the final hour, DMEM/F12 media was used, unless stated otherwise. The cells were collected for RNA extraction and cell culture supernatant was used to analyze concentrations of cAMP, IL-1β, IL-18, TNF-α, and caspase-1 using ELISA.
Measurement of microglial Ca++ response
Adult mice microglial cells isolated from adult B6, GPCR19-/-, and P2X7R-/- mice were seeded on 25 mm cover glasses (Deckglaser) in a 6 well plate. After 48 h culture with DMEM/F12 media containing 20% FBS and 10 ng/ml M-CSF in a 95% air and 5% CO2 atmosphere at 37°C, cells with glass coverslips were transferred and loaded with 2 µM Fluo-4/AM for 30 min at 37°C in a physiological external solution consisting of 138 mM NaCl, 5.6 mM KCl, 0.5 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4). After loading, cells on the coverslips were transferred to an open perfusion chamber and fluorescence was measured at 494/506 nm using a fluorescence microscope (Nikon, Tokyo, Japan). The microscope was equipped with an LED lamp (Andover, UK), integrated shutter, and cooled EM-CCD camera. The camera and shutter were controlled using MetaMorph software (Molecular Devices, Foster City, CA). Single cells were defined as regions of interest (ROIs). The 16-bit grayscale images with a binning of 1 x 1 were captured every 1 s with a ranging exposure time. Data were processed using OriginPro 8 software (OriginLab) and merged from three independent experiments.
BV2 cells were seeded on 25 mm cover glasses in a 6 well plate. After starvation, cells were treated with Aβ (2 µM) with or without TDCA (400 ng/ml) for 24 h. Cells with glass coverslips were transferred and loaded with 2 µM Fluo-4/AM for 30 min at 37°C in a physiological external solution mentioned above. After loading, cells were transferred to an open perfusion chamber and fluorescence was measured at 494/506 nm as previously described.
BV2 cell Ca++ sensing was measured using a BD calcium assay kit (BD bioscience, San Diego, CA, USA) according to the manufacturer’s protocol. In a 6 well plate, 1.5 x 105 cells/2 ml media were treated with Aβ with or without TDCA for 24 h after serum starvation. Cells were harvested in a FACS tube from the cell culture plate, washed with complete RPMI media, loaded with loading dye, and incubated for 1 h at 37°C in a 5% CO2 atmosphere. Cells were acquired for 1 min for basal signaling, then incubated with BzATP (300 µM) for 2 min, and recorded for an additional 4 min using flow cytometry (LSRFortessa, BD Biosciences, San Jose, CA, USA). Relative Fluorescent Units (RFU) were calculated using FlowJo version 9.0 (Treestar, Ashland, OR, USA).
Immunoblot
The cortex was harvested from B6, 5xFAD, GPCR19-/-, and P2X7R-/- mice, lysed with RIPA buffer (Thermo Fisher Scientific, Meridian Road, Rockford, USA) supplemented with phosphatase inhibitor cocktail 2 (Sigma-Aldrich), and centrifuged to remove cell debris. The concentrations of the prepared protein samples were determined using a BCA kit (Thermo Fisher Scientific). Protein samples were separated by electrophoresis on 10% sodium dodecyl sulfate–polyacrylamide gels and then transferred electrophoretically to Immobilon, polyvinylidene difluoride membranes (Merck, Millipore, Billerica, MA, USA). The membranes were incubated at 4°C overnight with anti-GPCR19 polyclonal antibody (Novus Biologicals, Littleton, CO, USA), P2X7R monoclonal antibody (P2X7R (Clone 1F11, Biolegend, San Diego, CA, USA), and anti‐β‐actin (Sigma-Aldrich). The following day, the membranes were washed and then incubated with horseradish peroxidase‐labeled anti‐rabbit or anti‐mouse secondary antibodies for 1 h at room temperature. Subsequently, membrane‐bound horseradish peroxidase‐labeled antibodies were detected using an enhanced chemiluminescence detection system, including the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). Densitometric quantification of the bands was conducted using ImageJ software (Rasband, W.S, NIH, Maryland, USA). Protein levels were normalized to β‐actin for quantification.
Immunocytochemistry
BV2 cells or primary microglia cells isolated from the adult mouse brain were seeded on 12 mm microscope cover glasses (Deckglaser) in 24 well plates. Cells were stained with anti-GPCR19 polyclonal antibody (Novus Biologicals) and P2X7R (Clone 1F11, Biolegend) with (cytoplasmic staining) or without permeabilization (surface staining) at 4°C overnight, followed by staining with goat anti-rabbit IgG, Alexa Fluor 488 (Thermo Fisher Scientific), and goat anti-rat IgG, Alexa Fluor 546 (Invitrogen) for 1 h at RT.
To stain inflammasomal components, BV2 or primary microglia cells isolated from the brains of one~two-day-old B6 mice were seeded on 12 mm microscope cover glasses in 24 well plates, and were treated with Aβ (2 µM), TDCA (400 ng/ml) for 24 h, and ATP (1 mM) for the final hour. The cells were permeabilized and stained with anti-NLRP3 polyclonal antibody (Abcam, Cambridge, United Kingdom) and anti-ASC antibody (Clone B-3, Santa Cruz Biotechnology, Inc. Dallas, Texas, USA) at 4°C overnight, followed by staining with secondary polyclonal antibodies, Alexa Flour 488-labeled goat anti-rabbit IgG, or Alexa Flour 532-labeled goat anti-mouse IgG (Invitrogen) for 1 h at RT.
To stain intracellular GPCR19 and cell surface CD47 and SRA, BV2 cells were treated with Aβ (2 µM) and TDCA (400 ng/ml) for 24 h. GPCR19 polyclonal antibody (Novus Biologicals), CD47 (GeneTex, Alton Pkwy Irvine, CA, USA), and CD204-SRA (Thermo Fisher Scientific) were used separately at 4°C overnight, followed by staining with goat anti-rabbit IgG, Alexa Fluor 488 (Thermo Fisher Scientific) for 1 h at RT.
For cytoplasmic staining, cells were treated with 0.3% Triton X-100 (Sigma-Aldrich, Steinheim, Germany) for 30 min. Control normal rabbit IgG (CST, Danvers, MA, USA), normal mouse IgG (Santa Cruz Biotechnology), or purified rat IgG (Biolegend) were used as experimental controls. After rinsing secondary polyclonal antibodies, cover glasses were placed on DAPI mounting solution (Vector laboratories, Burlingam, CA, USA) on glass slides. Fluorescence imaging was performed using Confocal Microscope A1 (Nikon, ECLIPSE Ti, New York, USA). The % co-localization of NLRP3-ASC was measured using ImageJ software after setting the color threshold caliper on the only yellow color. NIS-Elements.AR.Ink (version 4.2, Nikon) was used to measure mean fluorescence intensity (MFI) of ROI.
Quantitative RT-PCR
Cells were harvested after treatment and total RNA was isolated using a RNeasy Plus Mini kit (QIAGEN, Hilden, Germany). cDNA was prepared from 1 µg of total RNA using a Maxime RT PreMix kit (iNtRON Biotechnology, Gyeonggi-do, South Korea). Thereafter, real-time quantitative PCR (qPCR) was performed using SYBR Green Fast mix (Applied Biosystems, Woolston, Warrington, UK) and primers specific to target genes (Supplementary Table 1) in the StepOnePlusTM Real-Time PCR system (Applied Biosystems, Marsiling Industrial Estate Road, Singapore). Expression levels of target mRNAs were analyzed using the ddCt method and were normalized to expression levels of mouse GAPDH, a housekeeping gene used as an endogenous control. All fold changes are expressed relative to the control group.
ELISA
Cell secreted cytokines were measured from cell culture supernatant using commercially available ELISA kits for Mouse IL-6, TNF-α, and IL-1β/IL-1F2 (R&D Systems, Minneapolis, MN, USA), IL-18 (MBL, Naka-Ku, Nagoya Aichi, Japan), and Caspase-1 (Novus Biologicals) according to the protocol supplied by the manufacturer. BV2 cells were cultured in the presence of Aβ (2 µM), TDCA (400 ng/ml), or KH7 (4 μΜ) for 24 h. Cells were harvested and lysed with 0.1 M HCl to measure intracellular cAMP using a cAMP Assay Kit (Abcam) according to the manufacturer’s protocol.
Measurement of ROS production
BV2 cells were harvested in a FACS tube after treatment with Aβ (1~4 µM) and TDCA (400 ng/ml) for 24 h. Cells were washed with DPBS and incubated with 5 μM of 2′-7′-dichlorofluorescin diacetate (Invitrogen, Eugene, Oregon, US) in DMEM containing 1% penicillin and streptomycin for 30 min at room temperature in the dark. Samples were washed in DPBS, suspended in FACS buffer containing DAPI (0.3 µg/ml), then immediately acquired and analyzed using a BD LSR Fortessa flow cytometer (BD Bioscience, San Jose, CA, USA) and FlowJo v9 software.
Phagocytosis of Fluorescent Aβ oligomer
BV2 cells were seeded on microscope cover glasses in 24 well plates for confocal microscopy. Cells were treated with or without TDCA for 12 h. HilyteFluor™488 labeled Aβ (1-42) (AnaSpec, Freemont, CA, USA) were used for fluorescent Aβ oligomers as mentioned above. After pre-treatment with TDCA cells were incubated with fluorescent Aβ oligomers for 3 h, fixed with 4% paraformaldehyde (PFA), and permeabilized with 0.3% Triton X-100. After blocking, cells were incubated at 4°C overnight with primary antibody for LAMP2 (Clone M3/84, BD Pharmingen Inc, San Jose, CA, USA) or control rat IgG. After rinsing, cells were incubated with goat anti-Rat IgG (H+L) secondary Antibody (Invitrogen) for 1 h at room temperature in the dark. After rinsing, cover glasses were placed on the DAPI mounting solution on the glass slide. Fluorescence imaging was performed using Confocal Microscope A1. The % co-localization of fAβ-LAMP2 was measured using ImageJ software after setting the color threshold caliper on the only yellow color.
Primary microglia isolated from one~two-day-old B6 mice were cultured, pre-treated with TDCA (400 ng/ml) for 12 h and then incubated with fluorescent Aβ (1–42) oligomer (green) for 3 h. After incubation with f-Aβ, cells were washed with ice cold PBS and fixed with 4% paraformaldehyde (PFA). After blocking, cells were incubated at 4°C overnight with a primary antibody for P2X7R (Biolegend) or control IgG. Cells were then rinsed and incubated with Goat anti-Mouse IgG (H+L) Secondary Antibody (Invitrogen) for 1 h at room temperature in the dark. After rinsing, cover glasses were placed on the DAPI mounting solution on the glass slide. Fluorescence imaging was performed using Confocal Microscope A1. NIS-Elements.AR.Ink (version 4.2, Nikon) was used to measure mean fluorescence intensity (MFI) of ROI.
Animals
The 5xFAD mice co-overexpress high levels of APP with three FAD mutations (Swedish (K670N/M671L), Florida (I716V), and London (V717I)), and high levels of presenilins 1 (PSEN1) with two FAD mutations (M146L, L286V), which are specifically overexpressed in the brain, regulated by neural-specific Thy1 promoter51. 5xFAD mice were maintained by breeding male 5xFAD mice with female B6 mice. The SJL F1 hybrid was produced by an SJL male and a B6 female. PCR was performed for genotyping of the mice. 5xFAD or SJL mice were kindly provided by Professor Mook-Jung, In-hee or Professor Sung, Jung-Joon, respectively, of Seoul National University. All animal experiments were approved by the institutional animal care and use committees (IACUC) of Seoul National University (SNU-170517-25) and performed in accordance with animal ethics regulations. Mice were maintained in specific pathogen-free conditions at the animal facility of Wide River Institute of Immunology.
Drug administration
5xFAD male and female mice (8 to 10 weeks old) were injected intraperitoneally (i.p.) with either 1 mg/kg of TDCA or PBS five times/week for ten weeks. Age and gender matched non-transgenic littermates were used as a control group. Behavioral tests were performed after treatment and then mice were sacrificed for further experimentation. In the case of oral (p.o.) treatment, 5 mg/kg or 10 mg/kg TDCA was mixed with Monoolein (1-Oleoyal-rac-glucerol) and Oleic acid (TDCA: Monoolein: Oleic acid = 1: 2: 1) in PBS. TDCA (5 or 10 mg/kg) or PBS p.o. were administered using gastric gavage five/week for 14 weeks. Behavioral tests were performed during the last two~three weeks of treatment.
Morris water maze test
The maze was composed of a circular pool (1.5 m in diameter, 80 cm in height) with spatial cues at three different locations. Before testing, the pool was filled with opaque water adjusted to 20 ± 1°C. On the first day, mice were allowed to freely swim in the water for 60 seconds to find the escape platform located in one quadrant of the pool. When they failed to find the platform, the mice were guided to the platform. Once on the platform, mice were allowed to remain there for 30 sec. From the next day for four consecutive days, the same procedure was repeated from three different starting points to train the mouse, and the time to reach the platform was recorded every day. After the four-day training period, the probe test was performed in the same manner, but without the platform. Each mouse was allowed to swim from one starting point for 60 seconds, which was recorded using a video camera. The video was analyzed for the movement of mice in the water using tracking software (SMART3.0, Panlab Harvard Apparatus, Barcelona, Spain) to count the number of crossings, the time on the platform, and to measure the time spent at each quadrant of the pool.
Y-maze test
Mouse functional behavior tests were performed on TDCA- or PBS-treated 5xFAD mice and WT (B6) control mice. The Y-maze test was assessed over the course of four days. On the first two days, individual mice were habituated to the task room and experimenter for 5 min. On the third day after task room and experimenter habituation, mice were allowed to habituate to the Y-maze for less than 1 min. On the last day, mice entered the middle of the Y-maze and were allowed to move freely within the maze for 8 min. Each mouse movement was recorded using a video camera. The video was analyzed for all mouse entries regarding limbs that pass through each half arm of the maze. Total arm entries and percentage of alteration were counted for each mouse and compared between groups of mice.
Novel object recognition test (NORT)
NORT was assessed over the course of four days. The apparatus consisted of a white acrylic box (350 mm x 450 mm x 250 mm). The basement of the box was divided into 6 equal rectangles. On the first two days, each mouse was habituated to the box for 10 min. On the third day, two similar cylindrical objects were fixed in the box and mice were allowed to explore the objects freely for 10 min. On the last day, one cylindrical object was replaced by a similar size different object and the mouse explored this for 5 min. Exploration of two cylindrical objects on the third day, and exploration of the novel object and cylindrical object on day four was recorded for each mouse using a video camera. Total time and frequency of novel and old object exploration were counted for each mouse using the video footage. The percent discrimination index of the novel object was calculated from exploration time of novel and old object.
Flow cytometry analysis of brain and spleen cells
Cells from the isolated brain and spleen were prepared from TDCA- or PBS-administered mice after ten weeks of i.p. administration. In brief, mice were euthanized using Isoflurane (Hana Pharmaceutical, Korea) and cardiac perfused with ice-cold Dulbecco’s PBS (Welgene; LB001-02). Brain cells were prepared using the Adult Brain Dissociation Kit, mouse and rat (Miltenyi Biotec GmbH) using the GentleMACS dissociator (Miltenyi Biotec GmbH) according to the manufacturer’s protocol. A single cell suspension was obtained by passing the cell through a 70 µm cell strainer (MACS SmartStrainers, Miltenyi Biotec GmbH) and myelin was depleted using the percoll solution supplied with the Brain Dissociation Kit. Splenocytes were prepared by simply mincing and passing the whole spleen through a 40 μM nylon filter (BD Falcon, Bedford, MA). For lysis of red blood cells from splenocytes, cells were incubated with 2 ml of ammonium-chloride-potassium lysing buffer for 2 min. Cells were suspended in the FACS buffer: DPBS containing 1% FBS and 1 mM EDTA (Invitrogen). Cells were incubated with 2.5 ng purified anti-mouse CD16/32 mAb (Clone 2.4G2, BD Pharmingen) for 107 cells for 30 min on ice. Cells were stained with fluorochrome conjugated monoclonal antibodies: CD45-PerCP (Clone 30-F11), CD11b-APCcy7 (Clone: M1/70), Ly6C-FITC (Clone: AL21), and Ly6G-PE (Clone: 1A8, all obtained from BD Pharmingen) for 30 min at 4°C. Samples were washed in DPBS, suspended in a FACS buffer containing DAPI (0.3 µg/ml), and immediately acquired using flow cytometry (LSRFortessa, BD Biosciences). Further analysis was performed using FlowJo version 9.0. Total numbers of live brain cells and splenocytes were counted after Trypan blue staining, and cell subpopulations were calculated based on the % of total cells after FlowJo analysis.
Primary microglia isolated from one~two-day-old B6 mice were cultured and treated with Aβ (2 µM) and TDCA (400 ng/ml) for 48 h. Cells were harvested and incubated with CD86-PE-cy7 (Clone: GL1, BD Pharmingen) and CD206-PE (Clone: C068C2, Biolegend) for 30 min at 4°C. Samples were washed in DPBS, suspended in a FACS buffer containing DAPI (0.3 µg/ml), and immediately acquired using flow cytometry. Further analysis was performed using FlowJo.
Immunohistochemistry
TDCA- or PBS-treated 5xFAD or B6 mice were anesthetized and perfused with ice-cold PBS. Brains were harvested and maintained in 10% neutral buffer formalin (Sigma-Aldrich) for 24 h at 4°C and then embedded in paraffin (Lecia, Illinois, USA). The paraffin embedded brains were cut (3 μΜ) using microtome (Thermo Fisher Scientific), then deparaffinized in xylene, and rehydrated in a graded ethanol series (100 %, 90%, 80%, and 70%). Antigen unmasking was performed by heating the brain sections in citrate-based buffer (pH 6, Vector laboratories, Burlingam, CA, USA). The sections were incubated in 0.3% Triton X-100 for 30 min at room temperature for intracellular staining, and then blocked with blocking solution of 10% normal goat serum (Thermo Fisher Scientific) and 1% BSA in PBS for 1 h. Tissue samples were incubated overnight at 4°C with the following primary antibodies: GPCR19 (Novus Biologicals), NLRP3 (Abcam), ASC (Santa Cruz Biotechnology), NeuN (Clone A60, Merk Millipore, Temecula, CA, USA), Iba-1 (Wako, Osaka, Japan), and GFAP (Thermo Fisher Scientific). Slices were subsequently incubated for 1 h at room temperature with Alexa 488, 532, or 546-conjugated IgG secondary antibodies, as appropriate, then counterstained with DAPI for 10 min
The Aβ core plaque was labeled by treating tissue with 1% Thioflavin-S (Sigma-Aldrich) in PBS for 10 min at room temperature after being deparaffinized in xylene and rehydrated in a graded ethanol series (100 %, 90%, 80%, and 70%). The tissue slides were then washed thrice with 70% ethanol following DW three times. Fluorescence imaging was performed using Confocal Microscope A1 (Nikon). NIS-Elements.AR.Ink (version 4.2, Nikon) was used to measure mean fluorescence intensity (MFI) of ROI.
Global protein profiling
Anesthetized Mice were perfused with cold PBS and the whole brain was extracted, followed by snap-freezing using liquid nitrogen. The frontal cortex and hippocampus region of each mouse was collected and digested using urea (8 M)-based in-solution digestion. After protein quantification using the BCA assay (Micro BCA Protein Assay Kit, Thermo Fisher Scientific, Bremen, Germany), samples were pooled together group by group and 300 µg of tissue extracts per group were separated via high pH reversed-phase fractionation using an Agilent 1260 HPLC infinity purification system (Agilent Technology, Santa Clara, CA, USA). The fractionated peptide samples were subsequently loaded onto traps (C18, 3 μm, 0.7 cm, Thermo Fisher Scientific) and EASY-Spray columns (C18, 2 µm, 100 Å, 50 cm, Thermo Fisher Scientific). Easy nano II Ultra Performance Liquid Chromatography and Q-Exactive Mass Spectrometry systems (Thermo Fisher Scientific) were used to separate the peptides. For Q-Exactive, a top 10 method was used. The Orbitrap mass analyzer was used to acquire full MS scans (m/z 300 - 1,600 range; resolution, 70,000). The Trans Proteomic Pipeline (Seattle Proteomic Center, Seattle, WA, USA) was used to convert the mass data files into mzXML files. Peptide masses were searched using a concatenated forward and reverse mouse international protein index (IPI) database (decoy ipi.MOUSE.v3.80 database, 54285 entries)52 with the SEQUEST-Sorcerer platform (Thermo Fisher Scientific, Sage-N Research, Milpitas, CA). Sorcerer (Sage-N Research, Milpitas, CA, USA) was used to estimate the false discovery rate (FDR). Scaffold Q+ (Proteome Software, Portland, OR, USA) was used to compare spectral counts, validate MS/MS-based peptides, identify proteins (FDR < 1% in at least 2 peptides), and calculate Log2-fold changes (FC) and P-values (Student t-test). Differentially expressed proteins (FC >2, and P values <0.05) were uploaded to the web-based Ingenuity Pathway Analysis (IPA®) software (Version 26127183; QIAGEN) for functional analysis and protein interaction networks.
Statistical analysis
The data are expressed as the mean ± SEM and were analyzed using a two-sided, unpaired Student’s t test. The mean value between groups were compared using GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA, USA), unless otherwise indicated. P values < 0.05 were considered statistically significant.