119 4 Proteomic signature of macrophages cultured on collagen type 1 No functional metabolic differences between macrophages cultured on collagen- or uncoated plastic To investigate whether the higher expression of glycolytic proteins in macrophages cultured on collagen-coated substrates translated into changes in cellular metabolism, extracellular flux analysis was performed to assess glycolytic activity. No significant differences were found in glycolytic function between alveolar macrophages grown on collagen-coated or on uncoated wells (Figure 4a). Similarly, no differences in glycolytic function were found between alveolar macrophages cultured on either fibrous or globular collagen (Figure 4b). Furthermore, oxygen consumption rates were tracked and here too, no differences were found between macrophages cultured on plastic or macrophages cultured on either type of collagen (Supplementary Figure 2). As we found no differences in glycolytic function, we investigated whether the activity of four of the differentially expressed glycolytic enzymes was affected by culture conditions, i.e. hexokinase, phosphofructokinase, aldolase, and phosphoglycerate kinase. First, we again compared macrophages cultured on any type of collagen versus those cultured on uncoated plastic. We found that all enzyme activities tended to be lower in macrophages cultured on collagen compared to the uncoated control, but only the activity of aldolase was significantly lower (Figure 5a-d). We then investigated the effect of the collagen morphology on the activity of glycolytic enzymes and found that macrophages grown on fibrous collagen had significantly higher aldolase activity than macrophages grown on globular collagen (Figure 5e-h). Opposite trends between protein expression and protein activity were observed: when protein expression was higher, the activity was lower and vice versa (compare Figure 2c, 3b, 5a-d, and 5e-h).
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