Gene expression profiling of THP-1 cells on treatment with vitamin C
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Genotypic technology
PROVIDER: E-GEOD-73421 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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2014-05-07 | E-GEOD-57347 | biostudies-arrayexpress
Project description:We report a whole-genome expression profiling of PMA-treated THP-1 human macrophages as a host system, infected by W-Beijing strains of different sublineages or the H37Rv reference strain. Surprisingly, we found that host transcriptional responses were irrespective of inter-sublineage variations. Based on this finding, we further showed that such common host transcriptional responses were probably under the control of a regulatory network involving STATs, IRF-1, IRF-7 and Oct-1 transcriptional factors. We also suggested that these putative regulators might act in a cooperative manner to induce the expression of host-responsive genes, particularly those involved in cytokine-cytokine receptor interactions. THP-1 cells were infected by 11 different sublineages plus one reference strain H37Rv (two replications) at three time points (i.e., 4h, 18h and 48h). For each infection as well as a non-infection control, RNA was extracted using Trizol (Life Technologies)TRIZOL® reagent (Invitrogen), and subsequently amplified and labeled with biotin according to the standard Affymetrix® protocol. Specifically, RNA was DNase-treated and purified with RNeasy columns (Qiagen). The purified RNA was quantified using the ND-1000 Spectrophotometer (NanoDrop Technologies) and Agilent 2100 Bioanalyzer (Agilent Technologies). The biotinylated cRNA was then fragmented and subjected to hybridization on Human Genome U133 Plus 2.0 Array (Affymetrix, USA). The scanned images were converted to cell intensity files (CEL) using GeneChip® Operating Software (GCOS) (Affymetrix). These CEL raw expression data were normalized using Robust Multi-array Averaging (RMA) with quantile normalization in R (Bioconductor). Detection call-based filter was applied to remove all the probe-sets whose expression values were consistently below an empirically-determined value of minimum sensitivity, which were evaluated according to the 95th percentile of all the ‘Absent’ call-flagged signals of the entire dataset. Following the normalization and filtering, a gene expression matrix (termed as TB expression matrix) was constructed. The TB expression matrix was then subjected to component plane presentation integrated self-organizing map (CPP-SOM) for visual comparisons, or subjected to Cluster3.0/TreeView-1.0.8 softwares with Euclidean distance algorithm for sample classification of 13 time-course transcriptome profiles. Also, Linear Models for Microarray Data (LIMMA) bioconductor library[18] was applied to identify those differentially expressed genes between any two successive time points. The criteria for identifying the top significant genes for the designed contrast was based on P-values (< 0.01) corrected using Benjamini and Hochberg false discovery rate (FDR) procedure. Sets of genes of interest such as differentially expressed genes between 4h and 18h infections were uploaded onto Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 for identifying enriched gene ontology and KEGG pathways (FDR<0.01). For TFBS enrichment analysis, we applied PRIMA (PRomoter Integration in Microarray Analysis) program to identify TFs whose binding sites are significantly over-represented in a given gene set (i.e., differentially expressed genes) compared to background. Bonferroni-corrected P-values (< 0.01) were utilized to determine the significance of the binding site enrichments of TFs. Such computationally predicted TFs were considered as candidate regulators responsible for the putative co-regulations of differentially expressed genes.
2011-12-01 | E-GEOD-29628 | biostudies-arrayexpress
Project description:Endometriosis is an inflammatory disease and bone marrow-derived cells are abundant in endometriotic lesions and in the peritoneal fluid of women with the disease. This study tested the hypothesis that reciprocal communication occurs between macrophages and cultured human endometrial stromal cells and that this communication contributes to the pathology of endometriosis. Changes in gene expression elicited by exposure to factors secreted by the opposing cell type were measured by DNA microarray to test this hypothesis. 716 named genes were differentially expressed in cultured endometrial stromal cells in response to factors secreted by macrophages. Genes that were up-regulated included IL8/CXCL8, MMP3, phospholamban, CYR61/CCN1, CTGF/CCN2, tenascin C, and NNMT, whereas integrin alpha 6 was down-regulated. In contrast, 15 named genes were differentially expressed in macrophages in response to factors secreted by cultured endometrial stromal cells. 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Keywords: human, cultured endometrial stromal cell line, macrophage Experimental factors: hormone treatment and treatment with macrophage-conditioned medium of endometrial stromal cells, treatment of macrophages with endometrial stromal cell conditioned medium Experimental design: T HESC cells were grown in phenol red-free DMEM/F12 medium supplemented with 1% ITS+, 10% charcoal-dextran treated fetal bovine serum albumin, 1% pen/strep, and 500 ng/ml puromycin. THESC cells were treated for 8 days with vehicle (0.1% ethanol) or estradiol (10-8 M) plus progesterone (10-7 M medroxyprogesterone acetate). On day 6, serum was removed from the culture medium. On day 7, half of the cells were treated with U-937 macrophage-conditioned medium for 24 hours. The cells were harvested and RNA extracted on day 8. The experiment was repeated with three different passages of cells (passages 9, 11, and 13) to yield an experimental n of 3. The four groups of cells were vehicle-treated control, estradiol + progesterone, control + macrophage-conditioned medium, and estradiol + progesterone + macrophage-conditioned medium. The monocytic cell line, U-937, was maintained in suspension in RPMI-1640 with 10% fetal bovine serum and 1% pen/strep. The U-937 cells were treated with 32 nM phorbol myristate acetate (PMA) for 48 h to stimulate differentiation into macrophages. The U-937 macrophages were treated with THESC conditioned medium or with non-conditioned control medium. Total RNA was collected after 24 hours. The experiment was repeated with three different passages of cells (passages 7, 9, and 12) to yield an experimental n of 3. The two experimental groups were macrophages treated with non-conditioned control medium and macrophages treated with THESC-conditioned medium. Quality control steps: The cRNA that was synthesized from each cultured cell passage and treatment was used for hybridization to a single CodeLink (Applied Microarrays) whole human genome microarray. Only one sample was hybridized with each slide and only one dye (streptavidin-Alexa 647) was used so no dye swaps were necessary. Bacterial control spikes were used per manufacturer's instructions. Samples used, extract preparation and labelling: The origin of each biological sample: The samples were telomerase-immortalized human endometrial stromal cells and human U-937 monocytic cell line differentiation into macrophages by treatment with phorbol ester. The cell lines were obtained from American Type Culture Collection (#CRL-4003 and #CRL-1593.2). Manipulations of biological samples and protocols used: T HESC cells were grown in culture in phenol red-free DMEM/F12 medium supplemented with 1% ITS+, 2% charcoal/dextran treated fetal bovine serum, 1% penicillin/streptomycin, and 500 ng/ml puromycin. The cells were treated for 8 days with vehicle (0.1% ethanol), estradiol (10-8 M) plus progesterone (10-7 M medroxyprogesterone acetate), vehicle + macrophage-conditioned medium, or estradiol (10-8 M) + progesterone (medroxyprogesterone acetate 10-7 M) + macrophage-conditioned medium. On day 6, serum was removed from the culture medium of all cells. On day 7, half of the cells were treated with macrophage-conditioned medium for 24 hours. The cells were harvested and RNA extracted on day 8. The experiment was repeated with three different passages of cells (passages 9, 11, and 13) to yield an experimental n of 3. The four groups of cells were vehicle-treated control, estradiol + progesterone, control + macrophage-conditioned medium, and estradiol + progesterone + macrophage-conditioned medium. To prepare macrophage-conditioned medium, U-937 (ATCC #CRL-1593.2) cells were grown in RPMI-1640 medium with 10% fetal bovine serum until the cells covered 80% of the flask surface. These U-937 cells were treated with 32 nM PMA for 48 h to stimulate differentiation into macrophages. Differentiated macrophages were maintained in phenol red-free, fetal bovine serum-free DMEM/F12 medium supplemented with 1% penicillin/streptomycin for 48 hours. The cell suspension was centrifuged and the conditioned medium was collected, filtered, and stored at -80C. Before use, macrophage-conditioned medium was supplemented with 1% ITS+ and puromycin. The monocytic cell line, U-937, was maintained in suspension in RPMI-1640 with 10% fetal bovine serum and 1% pen/strep. The U-937 cells were treated with 32 nM PMA for 48 h to stimulate differentiation into macrophages. The differentiated macrophages were treated with THESC conditioned medium or with non-conditioned control medium. Total RNA was collected after 24 hours. Experimental factor: hormone treatment, treatment with macrophage-conditioned medium, and treatment with T-HESC cell conditioned medium Technical protocols: The cells were rinsed and TRI reagent (MRC) was added to the culture flasks. The cells were scraped into centrifuge tubes, bromochloropropane and sodium acetate were added, and the samples were centrifuged to separate the phases. The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen). The purity and quantity were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip. Labelled cRNA was prepared using the Message Amp II-Biotin Enhanced Kit from Ambion, following the manufacturer's Instruction Protocol. 1.0 microgram of T-HESC cell total RNA or 2 micrograms of macrophage total RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on spin columns included in the Ambion kit. The double-stranded cDNA was mixed with T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. 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