MRNA microarray analysis of gene changes in Huh7 cells treated with IFN-λ4, IFN-λ1 and IFN-α
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ABSTRACT: 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:Background: α-catulin may functions as an oncoprotein, sustaining proliferation by preventing cellular senescence and promoting cancer cell migration. In this study, we investigated the mechanism of α-catulin in cancer cell migration and metastasis in lung cancer. Method: α-catulin mRNA expression was isolated from A549/AS2neo (control) and A549/AS2neo-α-catulin stable cells. The Phalanx Human OneArray microarray analysis was performed to identify α-catulin downstream genes. Results: Overexpression of α-catulin increased cancer cell migration and metastasis. By using Phalanx Human OneArray microarray we have identified panel of genes altered by α-catulin overexpression, consisting of CDC42, intergrins and genes related to cytoskeleton remodeling. Conclusion: α-catulin is an oncoprotein and promotes lung cancer cell migration and metastasis. Two-condition experiment, Vector vs. alpha-catulin overexpression cells.The cDNAs encoding full-length human alpha-catulin were amplified and subcloned into lentiviral pLKO_AS2.neo which generated full-length alpha-catulin. Vector control or alpha-catulin lentivirus were transduced into A549 cells and G418 was used to select stable cells.
Project description:The source of IFN-γ in ovarian cancer microenvironment and its biological effect to the tumor cells is unclear. The immortalized human ovarian surface epithelial cell line, HOSE-E7/hTERT (HOSE) was treated with IFN-γ and expression microarray analysis was performed, and probes showing significantly higher values in IFN-γ-added group were termed â??IFN-γ signature genes (295 probes)â??. We then applied this signature to our ovarian cancer microarray data, which included 75 ovarian cancer clinical samples, by means of ss-GSEA. IFN-γ signature score was strongly correlated to the number of infiltrating CD4-positive or CD8-positive lymphocytes in the tumors. These data suggest that the IFN-γ in the ovarian cancer microenvironment is derived from lymphocytes, and an IFN-γ-rich microenvironment is strongly correlated to a lymphocyte-rich microenvironment. HOSE cells were incubated in 8 separate culture dishes, 4 dishes with and 4 dishes without 500 IU/ml recombinant human IFN-γ (R&D Systems) in the culture medium for 6 hours prior to the analysis.
Project description:An early hallmark of Toxoplasma infection is the rapid control of the parasite population by a potent multifaceted innate immune response that engages resident and homing immune cells along with pro- and counter-inflammatory cytokines. In this context, IFN-γ activates a variety of Toxoplasma-targeting activities in immune and non-immune cells, but can also contribute to host immune pathology. Toxoplasma has evolved mechanisms to timely counteract the host IFN-γ defenses by interfering with the transcription of IFN-γ-stimulated genes. We now have identified TgIST as a critical molecular switch that is secreted by intracellular parasites and traffics to the host cell nucleus where it inhibits STAT1-dependent proinflammatory gene expression. We show that TgIST not only sequesters STAT1 on dedicated loci but also promotes shaping of a nonpermissive chromatin through its capacity to recruit the NuRD transcriptional repressor. We found that during mice acute infection, TgIST-deficient parasites are rapidly eliminated by the homing Gr1(+) inflammatory monocytes thus highlighting the protective role of TgIST against IFN-γ-mediated killing. By uncovering TgIST functions, this study brings novel evidence on how Toxoplasma has devised a molecular weapon of choice to take control over a ubiquitous immune gene expression mechanism in metazoans, as a way to promote long-term parasitism. HFF, 2fTGH (STAT1+/+) and U3A (STAT1-/-) human cells were left uninfected or infected for 24 hours with 76KGFP and 76KGFPÎ?TgIST Toxoplasma strains and stimulated with 100 U/ml IFN-γ for 6 hours before gene expression was measured. Three independent experiments were performed for each condition.
Project description:Normal donor blood was incubated with or without IFN-g stimulation to establish an IFN-g gene signature. Systemic lupus erythematosus subjects were treated with placebo or AMG 811, a therapeutic anti-IFN--g antibody, and changes in the IFN--g signature in whole blood of these subjects was measured. Blood from healthy volunteers (n=4) was collected into sodium heparin tubes, and then untreated or treated with 294 pM recombinant human IFN-γ for 0, 24, or 48 hours with incubation at 37oC, 5 % CO2. The blood was then added to PAXgene tubes and processed for RNA purification. Twenty six subjects with stable, mild to moderate SLE were administered placebo or a single dose of AMG 811 ranging from 2 mg to 180 mg SC or 60 mg IV. Whole blood PAXgene tube samples were collected from all cohorts at baseline, day-1 (pre-dose), and at days 15, 56, and end of study (EOS) after treatment Arrays were hybridized in a Loop design.
Project description:Investigation of interacting effects of TB and IFN-g on gene expression regulation within human macrophage-like THP-1 cells To investigate the effect TB infection has on THP-1 macrophageâ??s response to IFN-g post-infection, in multiple experiments, THP-1 cells were exposed to each of the following four conditions: not treated, IFN-γ treated, M. tuberculosis infected and M. tuberculosis infected then IFN-γ treated. Total cellular RNA was then extracted. Recovered RNAs were pre-qualified for reproducibility by comparing qRT-PCR results of IFIT-2 and IP10 between replicate experiments. Four most reproducible replicate experiments were then selected for genome-wide mRNA abundance assays using Affymetrix microarray-based technology.
Project description:We examined the effect of acetaminophen (APAP) on the transcriptional response induced by interferon-β (IFN-β). Transcript levels from total RNA isolated rom murine livers were determined using G4121B whole genome arrays. Keywords: Pharmakinetics, drug treatment 6-8 week male CD-1 mice were administered vehicle alone, APAP, IFN-β or IFN-β + APAP (n=5 per treatment per time point 40 animals total) and were euthanized at 1.5 or 4 hours post-treatment. Agilent 22k whole genome arrays were used to measure changes in mRNA transcripts in the liver mRNA. Stratatagene Universal Mouse RNA was used as a reference.
Project description:Expression profiling of HCT116 colon cancer cells with different treatments. Illumina HumanHT-12 V4.0 expression beadchip was used to obtain expression profiles across more than 31,000 annotated genes. Total RNA obtained from HCT116 cells with different treatments, namely, cells with TNF-α (50ng/ml)/IFN-ɣ (50U/ml) treatment, cells with TNF-α (50 ng/ml)/IFN-ɣ (50 U/ml) and silibinin (100 uM) treatment and cells without any treatment. Each sample has 2 biological replicates.
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.
Project description:This dataset details the time-dependent response of human Huh7 hepatoma cells to type I and type III IFN. Despite activating similar signaling cascades, the type I and type III interferons (IFNs) differ in their ability to antagonize viral replication. However, it is not clear whether these cytokines induce unique antiviral states, particularly in the liver, where the clinically important hepatitis B and C viruses cause persistent infection. Here, microarray-based gene expression analysis is combined with mechanistic studies of signaling pathways to dynamically characterize the transcriptional responses induced by these cytokines in Huh7 hepatoma cells and primary human hepatocytes. Type I and III IFNs differed greatly in their level of interferon-stimulated gene (ISG) induction with a clearly detectable hierarchy (IFN-M-NM-2 > IFN-M-NM-1 > IFN-M-NM-;3 > IFN-M-NM-;1 > IFN-M-NM-;2). This hierarchy resulted in widely varying numbers of differentially expressed genes when quantified using common statistical thresholds, even though individual IFNs did not appear to regulate unique sets of genes. The kinetic profiles of IFN-induced gene expression were also qualitatively similar with the important exception of IFN-M-NM-1. While stimulation with either IFN-M-NM-2 or IFN-M-NM-;s resulted in a similar long-lasting ISG induction, IFN-M-NM-1 signaling peaked early after stimulation then declined due to a negative feedback mechanism. The quantitative expression hierarchy and unique kinetics of IFN-M-NM-1 suggest different roles for individual IFNs in the immune response, and help explain previously observed differences in antiviral activity. Huh7 cells were seeded into 6-well plates at the density of 3x10^5 cells/well and cultured overnight before stimulation with either 500 U/ml of IFN-M-NM-1 or IFN-M-NM-2, or 10 ng/ml of IFN-M-NM-;1, IFN-M-NM-;2, or IFN-M-NM-;3. Total RNA was harvested and isolated at 0.5, 1, 2, 4, 6, 12 and 24 hours post-incubation using RNeasy Mini Kit (Qiagen) following the Affymetrix GeneChip protocol of the Keck Affymetrix Resource facility at Yale University. IFN incubation at each time point was performed in duplicate. All subsequent processing, hybridization to the Illumina HumanHT-12 microarray, and quality control analyses were carried out by the Yale Center for Genome Analysis using standard protocols.
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.