76 Chapter 3 KDAC inhibition has only a minor effect on the metabolism of alveolar-like macrophages Since protein levels alone may not suffice to determine whether metabolic activity has changed upon LPS stimulation with or without KDAC inhibition, we assessed cellular metabolic rates using extracellular flux analysis. We first measured glycolytic function showing that LPS does not induce a glycolytic shift in alveolarlike macrophages, in accordance with results in primary alveolar macrophages by Woods et al (6) (Figure 6A-B). In addition, prior treatment with either KDAC3 or KDAC1/2/3 inhibitors did not affect glycolytic function, suggesting that the shift from pro- to anti-inflammatory behavior in these macrophages was not accompanied by a change in glycolytic activity (Figure 6). Figure 6: Effects of LPS and/or KDAC inhibitors MS275 or RGFP966 on glycolytic function of alveolar-like macrophages. Alveolar-like macrophages were incubated with 1 ug/ml KDAC1/2/3 inhibitor MS275, KDAC3 inhibitor RGFP966, or vehicle for 16 h and then stimulated with 10 ng/ml LPS or vehicle for 4 h. Extracellular acidification rates (ECAR) were measured with a glycolysis stress test using a Seahorse XF96 and normalized to protein concentration in each well using a BCA assay. (A) Macrophages were treated sequentially with glucose, oligomycin (ATP synthase inhibitor), and 2-DG (2-deoxyglucose, a glucose analogue). (B) Bars quantify glycolytic function. Data represent three biological replicates which are the average of 6 technical replicates per biological replicate. Colored dots indicate the different independent replicates. Glycolysis parameters were compared among treatments using a Wilcoxon test for CTRL vs LPS and Friedman test with a Dunn’s correction for multiple testing for LPS vs KDAC inhibitor+LPS. P<0.05 was considered significant.
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