Gene Expression Response to Interferon Treatment at 12h and 24 in A549 cells
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ABSTRACT: The goal of this experiment was to use global gene expression profiling to understand human lung epithelial cellular responses to interferon treatment. A549 cells were treated individually with IFN-α (10 ng ml−1; Sigma I4276), IFN-β (500 U ml−1; Sigma I4151) and IFN-γ (10 ng ml−1; Sigma I32265) or mock-treated. At 12 and 24 h post-treatment, cells were harvested (n = 3 wells per condition) and total RNA was extracted and analysed by microarray. Individual well replicates were pooled for each array by combining equal masses of total RNA (n=1 array per time point for each treatment and control).
Project description:To expand knowledge of the effects of interferon at the proteomic level, we treated HepG2 cells with IFN-alpha and IFN-lambda for 24 hours. HepG2.2.15 cells, a model for HBV infection, were also examined versus controls. MTT assays showed that optimized IFN levels (100 ng/ml) did not induce apoptosis relative to untreated controls. Including controls, more than 6,000 proteins were identified. Five replicates each of IFN-alpha treatment, IFN-lambda treatment, and control were performed, allowing confident identification of differentially expressed proteins. While a number of publications suggest that no interferon effect is evident upon HBV infection, our own results strongly suggest otherwise. Differential alterations of the proteasome were noted when comparing HBV infection against IFN-treatment. We also note that differential effects upon IFN treatment significantly overlapped with transcriptomic datasets when upregulation was examined. However, proteins downregulated upon IFN treatment show little overlap with these transcriptomic datasets.
Project description:A novel assay was developed for Daudi cells in which the antiviral (AV) and antiproliferative (AP) activities of interferon (IFN) can be measured simultaneously. Using this novel assay, conditions allowing IFN AV protection but no growth inhibition were identified and selected. Daudi cells were treated under these conditions, and gene expression microarray analyses were performed. The results of the analysis identified 25 genes associated with IFN-alpha AV activity. Upregulation of 23 IFN-induced genes was confirmed by using reverse transcription-PCR. Of 25 gene products, 17 were detected by Western blotting at 24 h. Of the 25 genes, 10 have not been previously linked to AV activity of IFN-alpha. The most upregulated gene was IFIT3 (for IFN-induced protein with tetratricopeptide repeats 3). The results from antibody neutralizing experiments suggested an association of the identified genes with IFN-alpha AV activity. This association was strengthened by results from IFIT3-small interfering RNA transfection experiments showing decreased expression of IFIT3 and a reduction in the AV activity induced by IFN-alpha. Overexpression of IFIT3 resulted in a decrease of virus titer. Transcription of AV genes after the treatment of cells with higher concentrations of IFN having an AP effect on Daudi cells suggested pleiotropic functions of identified gene products. Gene expression was initially measured in four Daudi cell groups (3 x 10(6) in 10 ml of RPMI) treated for 24 h at IFN concentrations selected to allow comparison of gene activity in AV activity environments with environments in which no AV activity was observed. These treatment groups were: (i) 0.0036 ng of IFN-alpha2c/ml (n=5) (allowing AV activity only); (ii) 0.00036 ng of IFN-alpha2c/ml (n=5) (no AV observed); (iii) 0.036 ng of HY-2/ml (n=3) (allowing AV activity); and (iv) 0.0036 ng of HY-2/ml (n=3) (no AV observed). To refine the list of genes associated with AV activity, samples showing AP phenotype (achieved by treatment with IFN-alpha2c [0.36 ng/ml] (n=3) or HY-2 [36 ng/ml]) (n=3) were compared to identically treated samples manipulated to display the AV phenotype through subsequent neutralization with anti-IFNAR1 MAb 64.10 (1 ug/ml) (n=6). Further analysis of gene expression profiles after 6 h of incubation helped indicate genes associated with early roles in IFN-induced AV mechanisms (n=6).
Project description:Proteomics of carboxylated polystyrene bead (1.0 um) phagosomes from murine bone marrow-derived macrophages. cells were either resting or treated with 100 U/ml IFN-γ (PeproTech) and 100 ng/ml LPS (Sigma) for 24 h, 20 ng/ml Interleukin-4 (IL4) (BD Pharmingen) for 48 h, 20 ng/ml Interleukin-13 (IL13), 10 ng/ml Interleukin-10 (IL10)for 48 h or Reprogrammed (IL4 was incubated with BMDMs for 24 h, and the medium was replaced with fresh medium containing IFN-γ/LPS to incubate for another 24 h). Phagosomes were isolated after 30 min bead inoculation.
Project description:The change of gene expression in Huh7 cells treated with IFN-λ4, IFN-λ1 and IFN-α was analyzed at 0h, 6h and 16h after different interferon treatments. Huh7 cells that had been treated with IFN-α (10 IU/ml), IFN-λ1 (20 ng/ml) and IFN-λ4 (20 ng/ml). Time points of 6 h and 16 h after stimulation were selected.
Project description:Melanomas are often infiltrated by activated inflammatory cells. Thus, melanoma cells are very likely stimulated by inflammatory cytokines. In order to assess the impact of common inflammatory cytokines, we investigated the gene expression profile of melanoma cell lines before and after cytokine treatment in vitro. Experiment Overall Design: 5 human melanoma cell lines were treated with either IFN-α 1,000 U/ml, IFN-γ 100 U/ml or TNF-α 10 ng/ml for 72 hours, or were left untreated. We analyzed their expression profile with Affymetrix expression arrays.
Project description:In this work we present an analytical strategy to systematically identify early regulators by combining gene regulatory networks (GRN) with GWAS. We hypothesized that early regulators in T-cell associated diseases could be found by defining upstream transcription factors (TFs) in T-cell differentiation. Time series expression and DNA methylation profiling of T-cell differentiation identified several upstream TFs, of which TFs involved in Th1/2 differentiation were most enriched for disease associated SNPs identified by GWAS. Naïve CD4+ T cells were isolated from buffy coat of four healthy donors using a naïve CD4+ T cell isolation kit (Miltenyi, Bergisch-Gladbach, Germany). Naïve CD4+ T cells were stimulated with plate-bound anti-CD3 (500 ng/mL), soluble anti-CD28 (500 ng/mL), in the presence of IL-12 (5 ng/mL), IL-2 (10 ng/mL) and antiâ??IL-4 (5 µg/mL) for Th1, IL-4 (10 ng/mL), IL-2 (10 ng/mL) and anti-IL-12 (5 µg/mL) and antiâ??IFN-g (5 µg/mL) for Th2. Cells were cultured for six days in Iscoveâ??s modified Dulbecco medium (IMDM) supplemented with 2 mM L-glutamine (PAA Laboratories, Linz, Austria), 10% heat-inactivated FCS (PAA Laboratories, Linz, Austria), 5 µM βâ??mercaptoethanol (Sigma-Aldrich, St. Louis, Missouri, USA) and 50 ug/mL gentamicin (Sigma-Aldrich, St. Louis, Missouri, USA). Cells were cultured for 6 days and then re-stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence of corresponding polarizing cytokines and antibodies for another 2 days (Zhang et al. 2013). RNA was extracted using a miRneasy Mini Kit (Qiagen). The RNA concentrations were analysed with NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). For the gene expression microarray analysis, The cRNA was prepared using a Low Input QuickAmp Labeling Kit. For Th1 and Th2 cells the gene expression microarray analysis was performed using SurePrint G3 Human Gene Expression 8x60K microarray kit, according to the manufactureâ??s instruction (Agilent Technologies).
Project description:Human monocyte THP-1 cells obtained from ATCC were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA) containing 10% FBS and supplemented with 10 mM Hepes (Gibco BRL). THP-1 was differentiated into macrophages by 24-h incubation with 160 nM phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO) followed by 24-h incubation in RPMI medium. Macrophages were further polarized to M1 macrophages by incubation with 10 pg/ml of lipopolysaccharide (LPS; Sigma) and 20 ng/ml of interferon (IFN)-γ (R&D Systems, MN) and are referred to as M(LPS+IFN-γ) cells. M2 macrophages were obtained by incubation with 20 ng/ml of interleukin (IL)-4 (R&D Systems) and are referred to as M(IL4) cells. To test the represented polarization marker of PMA differentiated-THP-1 macrophages stimulated with 20 ng ml(-1) IFNγ + 10 pg ml(-1) LPS and 20 ng ml(-1) IL-4, which are known to influence macrophage polarization in vetro into the M1 and M2 state, respectively. We used microarrays to detail the gene expression pools to identify distinct M1 and M2 state during this process.
Project description:Human retinal pigment epithelial (HRPE) cells in culture respond to inflammatory cytokines (IFN-? + TNF-? + IL-1? ) by increasing the expression of many cytokines and chemokines. The goal of this study was to delineate the role of miRNA in this process. We employed microarray analysis to study the effect of inflammatory cytokines on the miRNA expression in HRPE cells. HRPE cells were treated with a mixture of IFN-? (100 u), TNF-? (10 ng/ml) and IL-1? (10 ng/ml) for 16 hours, and the miRNA expression was analyzed using RNA preparations isolated from control and treated cells. The experiment was repeated. Control samples were labeled with Cy3 while the treated samples with Cy5.
Project description:Purpose of experiment was to compare transcriptomics of Calu-3 cells treated with either INF alpha or gamma. Calu-3 cells were treated with 1000 Units/ml of INFa (Sigma I4276) or 500 Units/ml of IFN γ (Sigma I3265). Cells were collected for RNA isolation at 0, 3, 6, and 18 or 22 h post treatment. Each treated sample was done in triplicate. (Triplicates are defined as 3 different wells, plated at the same time using the same cell stock for all replicates.)There are triplicate time-matched mock for each time point from the same cell stock as rest of samples. The NIAID Systems Virology Center
Project description:The islet primary non-function (PNF) is a serious problem in islet transplantation. In this study, we investigated whether DcR3-secreting transgenic (Tg) islets could reduce PNF. We generated transgenic mice expressing human DcR3. The transgenically expressed DcR3 protected islets from IFN-gama plus IL-1beta, or TNF-alpha plus IL-1beta-induced dysfunction and apoptosis in vitro. The Tg islets presented significantly reduced PNF after transplantation. <br><br> Three independent batches of islet isolation were carried out. For each batch, islets were obtained from 4 Tg and 4 WT mice, and pooled respectively. The pooled islets (Tg or WT) were then divided into 4 groups: 2 were cultured in the presence of IFN-gamma (0.5 ug/ml) plus IL-1beta (0.5 ng/ml) with 1 harvested at 24 h and 1 harvested at 48 h; 2 were cultured in the presence of TNF-alpha (100 ng/ml) plus IL-1beta (0.5 ng/ml) with 1 harvested at 24 h and 1 harvested at 48 h. Each treatment employed 3 chips using RNA from 3 different batches of islet isolation. For each treatment, genes with a mean signal strength difference above 2-fold between Tg and WT islets were selected for reverse PCR confirmation.