77 3 Responses of alveolar-like macrophages to lysine deacetylase inhibition We next assessed mitochondrial respiration in alveolar-like macrophages to investigate whether LPS-induced production of inflammatory mediators was fueled by oxidative phosphorylation and if so, whether this was affected by KDAC inhibitors. Treatment with only LPS did not change any of the mitochondrial respiration parameters compared to untreated controls (Figure 7). However, pretreatment with KDAC3 inhibitor RGFP966 resulted in a slightly higher spare capacity compared to treatment with LPS alone. Pretreatment with KDAC1/2/3 inhibitor MS275 did not affect mitochondrial respiration and neither did treatment with the KDAC inhibitors in absence of LPS (Supplementary Figure 2). Figure 7: Effects of LPS and/or KDAC inhibitors MS275 or RGFP966 on mitochondrial respiration in 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. Oxygen consumption rates (OCR) were measured with a mitochondrial stress test using Seahorse XF96 and normalized to protein concentration in each well using a BCA assay. (A) Macrophages were treated sequentially with oligomycin (ATP synthase inhibitor), FCCP (carbonyl cyanide-p- trifluoromethoxyphenyl-hydrazon), and rotenone. (B) Bars quantify mitochondrial respiration of macrophages treated with either LPS and/or KDAC inhibitors. Data represent seven biological replicates which is the average of 6 technical replicates per biological replicate. Colored dots indicate the different independent replicates. Mitochondrial parameters were compared among treatments using a Friedman with a Dunn’s correction for multiple testing for LPS vs KDACi+LPS. P<0.05 was considered significant.
RkJQdWJsaXNoZXIy MTk4NDMw