Transcriptional response of BMDMs to CRISPR KO of key lysosomal genes [HEX000527]
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE272357 | GEO | 2026/07/20
REPOSITORIES: GEO
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Project description:WT mouse BMDMs were treated with various drugs that impair different lysosomal properties for increasing durations and bulk RNA-seq was performed.
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Project description:This project explores the effects of Cdc42 gene-knock in effector cytotoxic T lymphocytes (CTLs). Effector CTLs were differentiated from OTI TCR transgenic mice for 5 days in vitro, then nucleofected with CRISPR/Cas9 RNPs targeting Cdc42 or non-targeting sequence.
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Project description:Purpose: In previous work, we found that iron supplementation to cells rescues impairment of cell viability and proliferation upon lysosomal dysfunction due to inhibition of the vATPase complex. The goal of this study is to characterize the transcriptomic changes (RNA-seq) upon Bafilomycin mediated lysosomal dysfunction with and without iron (Ferric Ammonium Citrate) supplementation. Methods: mRNA profiles of wild type HEK293T cells treated +/- ferric ammonium citrate (0.1mg/ml) and +/- BafilomycinA1 (10nM) were generated by deep sequencing, in triplicate, using Illumina NextSeq500. Results: We mapped about 30-40 million sequence reads per sample to the human genome (build GRCh38). Conclusions: Our study characterizes the transcriptomic changes upon lysosomal dysfunction upon small molecule (Bafilomycin A1) inhibition of vATPase complex. We conclude that there are several notable trasncriptomic changes upon lysosomal dysfunction some of which are reversed by iron supplementation.
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Project description:The CRISPR-Cas9 system enables efficient sequence-specific mutagenesis for creating germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-guideRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we established in vitro-assembled, fluorescent Cas9-sgRNA RNPs in stabilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. Sequence analysis of targeted loci in individual embryos reveals highly efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis reveals preliminary loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show efficient targeting of functional non-coding elements in gene-regulatory regions using saturating mutagenesis towards uncovering functional control elements in transgenic reporters and endogenous genes. Our results suggest that in vitro assembled, fluorescent Cas9-sgRNA RNPs provide a rapid reverse-genetics tool for direct and scalable loss-of-function studies beyond zebrafish applications.
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Project description:Reticuloendothelial macrophages engulf ~0.2 trillion senescent erythrocytes daily in a process called erythrophagocytosis (EP). This critical mechanism preserves systemic heme-iron homeostasis by regulating red blood cell (RBC) catabolism and iron recycling. Although extensive work has demonstrated the various effects on macrophage metabolic reprogramming by stimulation with proinflammatory cytokines, little is known about the impact of EP on the macrophage metabolome and proteome. Thus, we performed mass spectrometry-based metabolomics and proteomics analyses of bone marrow-derived macrophages (BMDMs) before and after EP of IgG-coated RBCs. Further, metabolomics was performed on BMDMs incubated with free IgG to ensure that changes to macrophage metabolism were due to opsonized RBCs and not to free IgG binding. Uniformly labeled tracing experiments were conducted on BMDMs in the presence and absence of IgG-coated RBCs to assess the flux of glucose through the pentose phosphate pathway (PPP). In this study, we demonstrate that EP significantly alters amino acid and fatty acid metabolism, the Krebs cycle, OXPHOS, and arachidonate-linoleate metabolism. Increases in levels of amino acids, lipids and oxylipins, heme products, and RBC-derived proteins are noted in BMDMs following EP. Tracing experiments with U-13C6 glucose indicated a slower flux through glycolysis and enhanced PPP activation. Notably, we show that it is fueled by glucose derived from the macrophages themselves or from the extracellular media prior to EP, but not from opsonized RBCs. The PPP-derived NADPH can then fuel the oxidative burst, leading to the generation of reactive oxygen species necessary to promote digestion of phagocytosed RBC proteins via radical attack. Results were confirmed by redox proteomics experiments, demonstrating the oxidation of Cys152 and Cys94 of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hemoglobin-β, respectively. Significant increases in early Krebs cycle and C5-branched dibasic acid metabolites (α-ketoglutarate and 2-hydroxyglutarate, respectively) indicate that EP promotes the dysregulation of mitochondrial metabolism. Lastly, EP stimulated aminolevulinic acid (ALA) synthase and arginase activity as indicated by significant accumulations of ALA and ornithine after IgG-mediated RBC ingestion. Importantly, EP-mediated metabolic reprogramming of BMDMs does not occur following exposure to IgG alone. In conclusion, we show that EP reprograms macrophage metabolism and modifies macrophage polarization.
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Project description:Lysosomal cathepsins regulate an exquisite range of biological functions, and their deregulation is associated with inflammatory, metabolic and degenerative disease in humans. Here, we identified a key cell-intrinsic role for cathepsin B as a negative feedback regulator of lysosomal biogenesis and autophagy. Mice and macrophages lacking cathepsin B activity had increased resistance to the cytosolic bacterial pathogen Francisella novicida. Genetic deletion or pharmacological inhibition of cathepsin B downregulated mTOR activity and prevented cleavage of the lysosomal calcium channel TRPML1. These events drove transcription of lysosomal and autophagy genes via the transcription factor TFEB, which increased lysosomal biogenesis and activation of autophagy-initiation kinase ULK1 for clearance of the bacteria. Our results identified a fundamental biological function of cathepsin B in providing a checkpoint for homeostatic maintenance of lysosome population and basic recycling functions in the cell. We used microarrays to explore the gene expression profiles differentially expressed in bone marrow-derived macrophages (BMDM) isolated from cathepsin B-/- and wild-type mice.
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Project description:We performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) assays in BMDMs, deciphered MG53's binding site.
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Project description:This experiement aims to know what the differences of protein translation are in the bone marrow derived macro-phages(BMDMs) from WT mice and Elp3 KO mice. We treated the BMDMs with or without IL-4 for 4 hours.
2022-09-12 | PXD033962 | Pride