Chemicals and reagents
LPS (from Escherichia coli), RPMI 1640 medium, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT), phosphate-buffered saline (PBS), antibiotics, L-glutamine and trypsin-EDTA were purchased from Gibco BRL (USA). Fetal bovine serum was purchased from Hyclone Laboratories Inc. (USA). Enzyme-linked immunosorbent assay (ELISA) test kits for Human IL-4 DuoSet and Human TNF-α DuoSet were obtained from R&D Systems (USA). Phospho-p38 MAPK (Thr180/Tyr182) rabbit monoclonal, phospho-SAPK/JNK (Thr183/Tyr185) rabbit monoclonal, phospho-ERK1/2 (Thr202/Tyr204) rabbit monoclonal, phospho- EIK-1 (Ser383) rabbit monoclonal, phospho-c-Fos (Ser32) rabbit monoclonal, phospho-c-Jun (Ser63) rabbit monoclonal, phospho-MMK3/MMK6 (Ser189/207) rabbit monoclonal, phospho-MAPKAPK-2 (Thr334) rabbit monoclonal, phospho-STAT1 (Try701) rabbit monoclonal, c-Fos rabbit monoclonal and c-Jun rabbit monoclonal antibodies were obtained from Cell Signaling Technology (USA). α-MG was provided by Dr. Iinuma Munekazu. A Bradford assay kit was purchased from Bio-Rad (Germany). A mirVana miRNA Isolation Kit was purchased from Ambion Inc. (USA). All other chemicals were purchased from Sigma-Aldrich (USA).
The human myeloid leukemic cell line U937 (ATCC No. CRL-1593.2) was purchased from the American Type Culture Collection (USA). The cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C under 5% CO2. EL4 cells were purchased from the American Type Culture Collection (ATCC TIB-39). The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C under 5% CO2.
U937 cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum, 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C under 5% CO2. For differentiation induction, the cells were seeded at a density of 2 × l07 cells per T150 flask. Differentiation was initiated by addition of phorbol-12-myristate-13-acetate to the culture medium to a final concentration of 50 ng/mL and allowed to proceed for 24 h. The U937 cells were then washed with complete culture media once and incubated in U937 culture medium for 48 h.
TNF-α and IL-4 cytokine assays
Differentiated U937 and EL-4 cells were aliquoted into the wells of 96-well plates at a density of 1.6 × 105 cells/well, mixed with different concentrations of α-MG. U937 cells were treated with 7.6, 12.5, 30.5 nM, respectively. EL-4 cells were treated with 3.0, 6.1, 12.2 nM, respectively and incubated for 30 min at 37°C under 5% CO2. LPS (0.1 ng/mL) was added to the differentiated U937 and EL-4 cells and incubated for 4 h. The culture media were then harvested for TNF-α analyses with Human TNF-α DuoSet ELISA kit (R&D Systems, USA). The culture media were harvested for IL-4 analyses with Human IL-4 DuoSet ELISA kit (R&D Systems, USA). The half maximal inhibitory concentration (IC50; μg/mL) was calculated with GraFit software (version 7; GraFit Data Analysis Software, USA).
Cytotoxicity assays were performed by the MTT method. Cells were incubated with 100 μL of 1 mg/mL MTT for 1 h at 37°C under 5% CO2. DMSO (100 μL) was added to dissolve the crystals and the OD560 was measured with an ELISA reader (Spectrafluor Plus, Tecan, Switzerland). The results were expressed as cell viability percentages among LPS-stimulated cells.
U937 cells were harvested into pellets, washed with PBS and stored at −80°C until extraction. Total RNA and small RNAs from the cultured cells were isolated using the mirVana miRNA Isolation Kit.
The gene expression profiles were analyzed using a whole human genome microarray containing 48,804 probes (BeadChip WG-6 version 3; Illumina Inc., USA). Biotin-labeled complementary RNA (cRNA) for hybridization was generated by in vitro transcription based on the Eberwine protocol using Illumina Human Whole Genome-6 expression BeadChip kits (Illumina Inc, USA). Total RNA (500 ng) was reverse-transcribed into cDNA, followed by linear amplification steps according to an Illumina TotalPrep RNA Amplification Kit (Ambion Inc., USA). Hybridization was performed with 1.5 μg of biotin-labeled cRNA in each BeadChip WG-6 array. After incubation at 58°C for 16 h, the BeadChip WG-6 was washed with fresh wash tray according to Illumina Whohle-Genome Gene Expression Direct Hybridization Assay, stained with streptavidin-Cy3 dye (Amersham Biosciences, Buckinghamshire, UK) and scanned as described in the Illumina manual. The HumanWG-6 v3.0 Expression BeadChip WG-6 contains six arrays on a single BeadChip WG-6, each with 48,804 probes derived from human genes in the NCBI RefSeq and UniGene databases. Each array on the BeadChip WG-6 covers genome-wide transcription of well-characterized genes, gene candidates and splice variants. The intensity of each probe was calculated as the average intensity of at least 15 beads. Array images and data output were processed using Illumina BeadStudio software (Ambion Inc, USA). The analysis methods for the gene expressions using R and BioConductor 2.10 Software Packages (Biobase, beadarray, limma packages of R/BioConductor were used).
Gene expression profiling
The gene expression profiles of undifferentiated and differentiated U937 cells were determined using the Illumina WG-6 version 3 Beadarray (Illumina Inc., USA). The raw intensity of spots was log-2 transformed for subsequent analysis. Quantile normalization was performed within all arrays to adjust the systematic variation of experiments and dye effects. Significantly changed genes were identified by Limma test with BH (Benjamini & Hochberg) adjust P values of less than 0.05.
Pathway and gene ontology analysis
The pathway and gene ontology analyses were performed using the MetaCore software (GeneGo Inc., USA), in which the differentially expressed gene sets for LPS and α-MG comprised the significantly changed genes between the two conditions and were annotated according to their biological processes based on gene ontology information.
Western blot analysis
Differentiated U937 cells at a density of 4 × 106 cells/well were pretreated with 13.4 nM α-MG for 30 min. The U937 culture medium contained 0.1 ng/mL LPS, and the incubation was continued for 4 h at 37°C under 5% CO2. The cells were washed twice with ice-cold PBS, resuspended in lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin, 1 mM PMSF) and centrifuged (Thermo Fisher Scientific Laboratory, USA) at 16,000 × g for 15 min at 4°C. The clarified cell lysates were used for Western blot analyses. The protein concentrations were determined using the Bradford assay kit (Ambion Inc., USA).
Protein extracts (20 μg) under reduced conditions were fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Hybond nitrocellulose membranes. The membranes were blocked with 3% non-fat milk in Tris-buffered saline containing 0.1% Tween for 1 h. The activations of p38, MAPK, JNK, ERK1/2, EIK-1, c-Fos, c-Jun, MAPK kinase 3 / MAPK kinase 6 (MMK3/MMK6), MAPK-activated protein kinase-2 (MAPKAPK-2) and signal transducers and activators of transcription-1 (STAT1) were assessed using phospho-p38 MAPK (Thr180/Tyr182) rabbit monoclonal, phospho-SAPK/JNK (Thr183/Tyr185) rabbit monoclonal, phospho-ERK1/2 (Thr202/Tyr204) rabbit monoclonal, phospho-EIK-1 (Ser383) rabbit monoclonal, phospho-c-Fos (Ser32) rabbit monoclonal, phospho-c-Jun (Ser63) rabbit monoclonal, phospho-MMK3/MMK6 (Ser189/207) rabbit monoclonal, phospho-MAPKAPK-2 (Thr334) rabbit monoclonal, phospho-STAT1 (Try701) rabbit monoclonal, c-Fos rabbit monoclonal and c-Jun rabbit monoclonal antibodies according to the manufacturer’s instructions. The antibody-bound protein bands were visualized by incubation with a horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich, USA), followed by detection using the ECL system (Amersham Pharmacia Biotech, USA). The integrated optical densities of the bands were quantified using Image J software (NIH, USA). Each sample was normalized by the β-tubulin content, as a constitutively expressed protein.
All experiments were performed in triplicate and repeated independently at least three times. Data were presented as mean ± standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA) using SAS 9.1.3 software (SAS Institute Inc., USA) followed by a Tukey test to determine any significant differences. P values less than 0.05 were considered statistically significant. Dose dependence was visually determined from the dose–response graphs.