ScRNAseq of CD45 enriched cells from spinal cords of Optn floxed and KO mice
Ontology highlight
ABSTRACT: We prepared spinal cords from Optn floxed and KO mice treated with vehicle or the RIPK1 inhibitor Nec-1s, which were enriched for CD45+ cells and single cell RNA sequenced using the inDrop protocol.
Project description:We prepared spinal cords from SOD1-G93A and WT mice treated with vehicle or the RIPK1 inhibitor Nec-1s, which were single cell RNA sequenced using the DropSeq protocol.
Project description:To determine the role of RIPK1 kinase-dependent transcriptional signaling in human fetal-derived astrocytes, we stimulated cells with the RIPK1 kinase-activating stimulus TNF/Smac/zVAD in the presence or absence of the RIPK1 kinase inhibitor Nec-1s. We identified various genes modulated in a RIPK1 kinase-dependent manner in human astrocytes, and the main biological pathways were related to an interferon signaling and anti-viral response.
Project description:To determine the role of RIPK1 kinase-dependent transcriptional signaling in microglia and astrocytes, we stimulated cells with RIPK1 kinase-activating stimuli TNF/5z-7 and TNF/Smac/zVAD in the presence or absence of the RIPK1 kinase inhibitor Nec-1s. We identified various genes modulated in a RIPK1 kinase-dependent manner with each stimulation in both microglia and astrocytes, and the main biological pathways were related to an inflammatory and immune response. We identified many cytokines and chemokines upregulated in both microglia and astrocytes upon RIPK1 kinase activation, demonstrating the contribution of RIPK1 kinase signaling to pro-inflammatory responses in microglia and astrocytes
Project description:We use a proximity labelling-based proteomics strategy (BioID) to map the interactome of OPTN. OPTN was tagged N and C-terminally with BirA* in RPE1 cells and then, after labelling with biotin, streptavidin pull downs were performed to identify OPTN-proximal proteins.
Project description:Gene level expression estimate using the Whole Transcript (WT) Assay approach of the Gene 1.0 ST Array System for Mouse. This assay was done to identify the RIPK1-dependent gene expression changes in mouse BMDMs. Cost-effective gene-level analysis based on whole-transcript coverage. We analyzed Bone Marrow Derived Macrophages (BMDMs) under 4 different conditions (Control, LPS, LPS/zVAD, LPS/zVAD/Nec-1) to assess inflammatory changes in RIPK1 kinase dependent manner compared to LPS, LPS/zVAD plus RIPK1 inhibitor Nec-1 and control.
Project description:By using Limited Proteolysis-Mass Spectrometry (LiP-MS) and Hydrogen Deuterium Exchange-Mass Spectrometry (HDX-MS) analysis, we identified the binding pocket of Nec-34 in RIPK1 kinase domain. This newly identified binding pocket of Nec-34 is distinct from distinct from that of Nec-1s.
Project description:Acetaminophen (APAP) is a leading cause of acute liver failure in Western countries result from the accumulation of damaged mitochondria. However, there is no related clinically useful therapeutic intervention. Optineurin (OPTN) is a multifunctional protein involved in autophagy, oxidative stress, as well as nuclear factor κB (NF-κB) and IRF3 signaling, and OPTN mutations are associated with several human diseases.Here, to study the role of OPTN in APAP induced liver injury, we performed RNA-seq analysis of isolated control and Optn-/- mouse liver tissues.