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Influence of murine norovirus on cell metabolism

ABSTRACT: 6.5 X 10^6 cells were plated in 10 cm Tissue Culture dishes and allowed to recover overnight at 37 degrees and 5% CO2. In the am, supernatant was removed, and 12 mL of medium containing either Murine Norovirus-1 (MNV) at an MOI=5 or medium containing a v/v match of mock lysate was added to the cells. Plates were rocked on ice for 1 hour, then cells were washed 3X with cold DPBS++, and plain medium as added to cells. Plates were then incubated for 7.5 hours at 37 degrees and 5% CO2. Cells were washed with 12 mL of 150 mM Ammonium Acetate, swirled 8 times, and immediately quenched with liquid nitrogen. Cells were then frozen at -80 degrees.

ORGANISM(S): Mouse Mus Musculus

TISSUE(S): Cultured Cells

SUBMITTER: Maureen Kachman

PROVIDER: ST000833 | MetabolomicsWorkbench | Wed Aug 02 00:00:00 BST 2017

REPOSITORIES: MetabolomicsWorkbench

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Project description:We present a microarray experiment with the same tet-inducible system but with multiple time points within 24 hr to capture early responses to Oct4 suppression. These data were analyzed together with published genome-wide ChIP data. We found that most tentative target genes (TTG) of Oct4 in ES cells were activated by Oct4 and only a few genes were suppressed. The same method was then applied to find target genes of Sox2 and Nanog based mostly on previously published data. Because the interaction between Oct4, Sox2, and Nanog is supported by immunoprecipitation, functional analysis, and co-localization of binding sites, we explored the relationships between their target genes. Keywords: time series design We used ZHBTc4 ES cells with a Tet-inducible Oct4 transgene. Cells were cultured for 2 passages on gelatin-coated plates in order to remove feeder cells and then transferred to gelatin-coated 6-well plates at the density of 1-2x105 cells/well and cultured in complete ES medium. Tetrocycline (TC) was added at 24 hr after cell plating, and then cells were harvested at 0hr (before adding TC), 3 hr, 6 hr, 12 hr, and 24 hr (2 replications each). RNA samples for later time points (24, 48, 72, 96, and 120 hr) were obtained from our earlier experiment with 3 replications. Two Nanog over-expressing clones were tested: (1) integrated transgene in MG1.19 parental cell line and (2) episomal transgene in EBRTcH3 parental line. EBRTcH3 and MG1.19 ESC lines were kind gifts of Dr. Hitoshi Niwa (RIKEN Center for Developmental Biology, Kobe, Japan). Both transgenic and parental cell lines were cultured for 2 passages on gelatin-coated plates and then transferred to gelatin-coated 6-well plates at the density of 1-2x105 cells/well and cultured for 3 days in 3 different conditions: (1) complete ES medium (see above); (2) complete medium without LIF, and (3) complete medium with 1 uM RA. Cells were cultured at 37 0C and 5% CO2 condition and the culture medium was changed daily. Over-expression of Nanog was evaluated by PCR. Total RNAs were extracted using TrizolTM (Invitrogen, USA) and Phase lock gelTM columns (Eppendorf/Brinkman) according to the manufacturer???s protocol. Total RNAs were precipitated with isopropanol, washed with 70% ethanol, and dissolved in DEPC-treated H2O. 2.5 ug of total RNA samples were labeled with Cy3-CTP using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent, USA). A reference target (Cy5-CTP-labeled) was prepared from the Universal Mouse Reference (UMR) RNA (Stratagene, USA).

Project description:Purpose: To characterize the processes defining the end dendritic cell (DC) phenotype, including the type of early transcriptional changes in pro-inflammatory cues towards regulatory or type 2 immune-based cues induced by a variety of exogenous and endogenous molecules. Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from human donors using Ficoll-Paque gradient centrifugation. CD14+ monocytes were isolated from PBMCs by magnetic-activated cell sorting (MACS) and cultured in complete medium consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 50 µM 2-Mercaptoethanol, 1% Penicillin-Streptomycin at 37°C and 5% CO2. During the differentiation stage, complete medium was added 150 U/mL recombinant human IL-4 and 160 U/mL recombinant human GM-CSF to obtain monocyte-derived dendritic cells (moDCs). Medium was replaced after 3 days of culturing, and moDCs were used 6 days after the start of culture. The cells were on average >70% CD1a-positive, and with <3% CD14+CD16+ cells, based on flow cytometry, and responded with high levels of IL-12p70 production upon stimulation with LPS and IFN-y (mean IL-12p70 production of 4641.8 pg/mL; SD = 688.1 pg/mL). Cells were harvested and rested for 1 hour at 37°C and 5% CO2 before stimulation. At this point, moDCs were supplemented with complete medium containing TSLP (to final concentration of 25 ng/mL), IL-25 (25 ng/mL), TSLP+IL-25 (both 25 ng/mL), helminth PCF (100 μg dry matter/mL, tested in different doses in pre-study experiments), butyrate (2 mM), or succinate (0.5 mM and 2 mM, di-sodium succinate hexahydrate). Unstimulated cells were supplemented with complete medium alone. Immediately afterwards, medium containing Escherichia coli O26:B6 LPS (final concentration of 100 ng/mL) and IFN-γ (10 ng/mL) was added. Cells were stimulated for 4 or 20 h at 37°C, 5% CO2, and 95% humidity. Results: Our data show that helminth PCF and butyrate treatment suppress the Th1-inducing pro-inflammatory DC phenotype through induction of different transcriptional programs in DCs. RNA sequencing indicated that helminth PCF treatment strongly inhibited the Th1 and Th17 polarizing ability of LPS+IFN-γ-matured DCs by downregulating myeloid differentiation primary response gene 88 (MyD88)-dependent and MyD88-independent pathways in TLR4 signaling. By contrast, butyrate treatment had a strong Th1-inhibiting action, while transcripts encoding important gut barrier defending factors such as IL18, IL1B and CXCL8 were upregulated. Conclusion: Collectively, our results further our understanding of how compounds from parasites and gut microbiota-derived butyrate may exert immunomodulatory effects on the host immune system.

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Influence of murine norovirus on cell metabolism

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