65 3 Responses of alveolar-like macrophages to lysine deacetylase inhibition 37 °C Collect media Substrate Cytokines secretion Centrifuge Add cold methanol/ water. Scrape cells and collect Rinse cells with PBS RNA isolation Centrifuge Interphase Polar phase Protein quantification Metabolome Scrape cells and collect Add cold chloroform Cell lysis Digestion Chromatography cDNA Sequencespecific primers Random primers Oligo(dT)s Genome and and and and Proteome Mass spectrometry ATP-linked respiration Proton leak Spare respiratory capacity Extracellular Flux analysis Using Seahorse Analyzers Mitochondrial stress test Maximal respiration Non-mitochondrial oxygen consumption Basal respiration Time (minutes) OCR (pmol/min/ μg) 0 5 10 15 0 20 40 80 60 Oligomycin FCCP Rotenone, Antimycin A Non-glycolytic acidification Glycolytic capacity Glycolytic reserve Glycolysis Time (minutes) ECAR (μpH/ min/μg) 0 0.5 1.0 1.5 2.0 0 20 40 80 60 Glucose Oligomycin 2-DG Glycolytic stress test Alveolar-like macrophages Figure 1: Representation of the workflow used to obtain different samples from alveolar-like macrophages. Depicted in red and represented by a pentagon are the steps necessary to measure cytokine secretion, in purple and represented by a triangle the steps necessary to measure gene expression, in green and represented by a circle the steps necessary to measure metabolite levels, in blue and represented by a square the steps necessary to measure protein expression, and in orange and represented by a star the steps necessary to perform extracellular flux analysis. Figure created with Biorender.com. Functional metabolic assays Macrophage glycolytic rates were measured using a Seahorse XFe96 Extracellular Flux Analyzer (Agilent, Santa Clara, USA). Macrophages were seeded at a density of 3.7 x 103/well in Seahorse XF96 cell culture microplates for 72 hours. Cells were then treated with KDAC inhibitors for 20 hours (MS275, RGFP966) and LPS was added for the last 4 hours of the experiment (KDAC inhibitor+LPS) or not (control). Cells were then equilibrated for 1 hour in the absence of CO2 at 37 °C with XF base media, which does not contain glucose but was complemented with glutamine
RkJQdWJsaXNoZXIy MTk4NDMw