113 4 Proteomic signature of macrophages cultured on collagen type 1 collagen morphology-specific effects, we also compared protein expression levels of macrophages cultured on fibrous or globular collagen. 142 proteins were found to be differentially expressed (Figure 1c and Supplementary Table 2) and 101 of these proteins were more than 1.25-fold upregulated in macrophages cultured on fibrous collagen, whereas only 19 proteins were downregulated (Supplementary table 2). Culturing macrophages on collagen results in higher expression of proteins involved in glycolysis Out of the 77 differentially expressed proteins between macrophages cultured on collagen type I and uncoated controls, 30 proteins showed a more than two-fold higher expression in collagen-coated conditions (Table 1). Three proteins even increased more than ten-fold: pigment epithelium-derived factor (Serpfin1), protein FAM118B (Fam118B), and guanylate-binding protein 4 (GBP4). Only the proteins caspase-9 (Casp9), Rac GTPase-activating protein 1 (Racgap1), and SOSS complex subunit B1 (Nabp2) were expressed at lower levels in macrophages cultured on collagen-coated samples (Table 1). Analysis of the biological processes in which the proteins that are upregulated by culturing on collagen type I are involved (Supplementary Table 1), indicated an effect on macrophage metabolism (Figure 2a). Pathway analysis revealed enrichment of the glycolysis pathway under collagen-coated conditions (Figure 2b). The presence of either fibrous or globular collagen induced a more than two-fold higher expression of liver type ATP-dependent 6-phosphofructokinase (Pfkl), a key enzyme in glycolysis. Additionally, the expression levels of two other members of the glycolysis pathway, fructose-biphosphate aldolase C (Aldoc) and phosphoglycerate kinase-1 (Pgk1) were higher in the presence of collagen type I (Figure 2c). An overview of the main enzymes involved in the glycolysis pathway can be found in Figure 3a.
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