Fig. 2

M1 macrophage secretome effects on human ASCs metabolism. A Representative confocal images of human ASCs cultured in differentially activated MQ-CM for 72 h. Cells were stained with CellMask Orange to visualize cell morphology. Scale bars indicate 200 µm. B Quantification of relative cell numbers in 72 h treated ASC. C ATP levels measured by ATP-Red and mitochondrial membrane potential (MMP) determined by TMRM staining (n = 6). Asterisks indicate p values of p<0.05 (*) and p<0.01 (**). D Energy profiling of MQ-CM treated ASCs at 72 h determined by Agilent Seahorse technology. The graph shows the correlation of mitochondrial respiration determined by oxygen consumption rate (OCR) and glycolysis measured by extracellular acidification rate (ECAR). Open squares indicate levels under basal conditions; closed squares indicate levels under oligomycin-induced mitochondrial uncoupling (stressed conditions) (n = 4). E Metabolic potential indicates the ability of cells to meet an energy demand via mitochondrial respiration and glycolysis. This is shown as the percentage increase of stressed OCR over baseline OCR and stressed ECAR over baseline ECAR (n = 4). F Representative immunoblots of MQ-CM treated ASC at 72 h analyzed for the expression of key regulatory enzymes of glycolysis. G Representative immunoblots of MQ-CM-treated ASCs showing levels and activation of intracellular kinases over time. All data are shown as mean ± SEM