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Summary: Polyunsaturated fatty acids (PUFAs) and their oxidized products (oxylipins) are important mediators in intra- and extra-cellular signaling. We describe here the simultaneous quantification of 163 PUFAs and oxylipins using liquid chromatography-mass spectrometry (LC-MS). The protocol details steps for PUFA purification from various biological materials, the conditions for LC-MS analysis, as well as quantitative approaches for data evaluation. We provide an example of PUFA quantification in animal tissue along with the bioinformatic protocol, enabling efficient inter-sample comparison and statistical analysis.For complete details on the use and execution of this protocol, please refer to Vila et al.,1 Costanza et al.,2 Blomme et al.,3 and Blomme et al.4 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in several cellular processes including pathogen recognition and inflammatory responses. We describe a protocol to activate the cGAS-STING pathway in murine cells using nucleic acids transfection. We describe how to prepare the nucleic acid probes and validate activation of the pathway by western blot and gene expression analysis. The protocol can be applied to investigate cGAS-STING signaling in both murine and human cell lines.For complete details on the use and execution of this protocol, please refer to Vila et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Adipose tissue fibrosis development blocks adipocyte hypertrophy and favors ectopic lipid accumulation. Here, we show that adipose tissue fibrosis is associated with obesity and insulin resistance in humans and mice. Kinetic studies in C3H mice fed a high-fat diet show activation of macrophages and progression of fibrosis along with adipocyte metabolic dysfunction and death. Adipose tissue fibrosis is attenuated by macrophage depletion. Impairment of Toll-like receptor 4 signaling protects mice from obesity-induced fibrosis. The presence of a functional Toll-like receptor 4 on adipose tissue hematopoietic cells is necessary for the initiation of adipose tissue fibrosis. Continuous low-dose infusion of the Toll-like receptor 4 ligand, lipopolysaccharide, promotes adipose tissue fibrosis. Ex vivo, lipopolysaccharide-mediated induction of fibrosis is prevented by antibodies against the profibrotic factor TGFβ1. Together, these results indicate that obesity and endotoxemia favor the development of adipose tissue fibrosis, a condition associated with insulin resistance, through immune cell Toll-like receptor 4.