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The effect of STING knockout on gene expression in DMXAA-induced BMDMs and BMDCs

ABSTRACT: The effect of STING knockout on gene expression in DMXAA-induced BMDMs and BMDCs

PROVIDER: PRJNA1057802 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR27364100_1.fastq.gz	Fastqsanger.gz
	SRR27364100_2.fastq.gz	Fastqsanger.gz
	SRR27364101_1.fastq.gz	Fastqsanger.gz
	SRR27364101_2.fastq.gz	Fastqsanger.gz
	SRR27364102_1.fastq.gz	Fastqsanger.gz

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The effect of STING knockout on gene expression in DMXAA-induced BMDMs and BMDCs

Project description:Myeloid-derived STING has been recognized to play a vital role in mediating the development of colitis. We here report that in BMDMs and BMDCs, knockout of STING inhibited pathways related to IL-12/23 production and IL-12 signaling, inflammation, and the maturation and activation of macrophages and DCs.

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Project description:BMDCs from WT, Fcgr2b-/- and Stinggt/gt were cultured, then stimulated with DMXAA for 3 and 6 hr. Cells were collected and lysed with 1 % IGEPAL CA-630, 0.5% TritonX-100, 150 mM NaCl, 50 mM Tris pH 7.4, 5% glycerol, 100 mM beta-Glycerophosphate, 2 mM Na3VO4 and 1X proteases inhibitor cocktail (Roche). First, antibody were mixed with the magnetic beads by adding 10 µg of STING antibody with 400 ug of SureBeads™ Protein A magnetic beads (Biorad, California, USA) and incubated for 1 hour at room temperature. Then, Protein lysates were added and incubated with antibody-conjugated beads for overnight at 4oC. After incubation, the beads were washed 3 times with wash buffer (150 mM NaCl and 50 mM Tris-HCL pH 7.4). Samples were eluted by adding 5X laemmli buffer and, boiled 950C for 10 minutes. The eluted protein samples were separated by 10 % SDS-PAGE gel. The STING interacting proteins from co-IP were analyzed by in-gel digestion followed by LC-MS/MS analysis.

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Project description:small RNAseq was preformed on Wt bone marrow-derived dendritic cells (BMDC) and miR-155 and miR-146a double knockout (DKO) BMDCs that received Wt exosomes to investigate the differences in transferred miRNA Small RNA profiles were generated from Wt donor BMDCs and DKO BMDCs given Wt exosomes 3 replicates in each group

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Project description:We report positional cloning and characterization of a novel Sting allele that fails to activate IFN production in the wild-derived mice of MOLF/Ei strain in response to HSV (Herpes Simplex Virus) and Listeria monocytogenes both in vitro and in vivo. We show that previously uncharacterized mutations in the N-terminal of STING are responsible for low levels of IFN due to failure of MOLF STING to respond to cytosolic STING agonists such as 2â3âcGAMP, 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) or poly(deoxyadenylic-deoxythymidylic) acid (dAdT). Using Next-Generation Sequencing (NGS) for the analysis of DNA-responses in congenic C57BL6.StingMOLF/MOLF (MOLF STING) mouse macrophages, we show that these mutations in MOLF/Ei discriminate in responses between different STING agonists. Macrophages from C57BL6.StingB6/B6Â¬ (B6 STING), or B6 STING expressing littermates were stimulated as a positive control for STING activation, and STING -/- (STING KO) macrophages served as a negative control. To examine differential gene regulation downstream of murine Tmem173 (STING) allelic variants. We stimulated macrophages from B6 mice congenic for MOLF STING; macrophages from B6 STING expressing littermates were stimulated as a positive control for STING activation, and STING -/- (STING KO) macrophages served as a negative control. One replicate of RNA-seq reads after each stimulation provided. Conditions are peritoneal macrophages from B6 congenic mice expressing: B6 STING, MOLF STING, or STING KO stimulated with 2â3âcGAMP, 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), poly(deoxyadenylic-deoxythymidylic) acid (dAdT), or no treatment. This constitutes a total of 12 reads analyzed in this data set.

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