72 Chapter 3 Figure 3: Effects of LPS and/or KDAC inhibitors MS275 or RGFP966 on SOCS3 and TGFβ mRNA expression in alveolar-like macrophages. Alveolar-like macrophages were incubated with 1 ug/ml (1 μM) KDAC1/2/3 inhibitor MS275 (KDAC1+2+3i), KDAC3 inhibitor RGFP966 (KDAC3i), or vehicle for 16 h and then stimulated with 10 ng/ml LPS or vehicle for 4 h. (A) Effects on SOCS3 mRNA expression. Colored dots indicate the different independent replicates. (B) Effects on TGFβ mRNA expression. A Wilcoxon test was used to compare control versus LPS stimulation. Colored dots indicate the different independent replicates. Groups stimulated with or without KDAC inhibitors were compared using a Friedman test with a Dunn’s correction for multiple testing (n=5). P<0.05 was considered significant. KDAC inhibition and metabolic reprogramming To understand the molecular mechanisms behind the anti-inflammatory effects of KDAC inhibitors we performed an untargeted proteome analysis on proteins isolated from macrophages (see Figure 1). We identified more than 4000 proteins and determined differentially expressed proteins between control and different treatment conditions. We identified 318 significantly upregulated and 85 downregulated proteins in cells treated with MS275+LPS compared to LPS (supplementary data, Supplementary Table 1). The proteins were considered upregulated if their fold change was higher than 1.5 and downregulated if the fold change was lower than 0.7 in combination with q-values lower than 0.05. The resulting list of proteins was used for pathway
RkJQdWJsaXNoZXIy MTk4NDMw