Figure 5
Effect of ABX464 on cytokine secretion and mRNA expression in BMDMs (a) Bone marrow isolated cells were cultured for 6 days in the presence of GM-CSF (50ng/ml) to differentiate into macrophages. Cells were kept in culture for additional 3 days in the presence of ABX464 (5 µM) or vehicle (DMSO) alone and for additional 6 hours stimulated with LPS (4 µg/ml). Cells were then kept in normal medium for additional 42 hours. Cell aliquots for RNA isolation and supernatants were taken at time 6, 12, 24 and 48 hours. (b) Culture supernatants of the indicated cell cultures were analyzed by CBA for the content of MCP-1, IL-6, TNFalpha and IL-10 as described in Materials and Methods. (c) A Venn diagram displaying the number of inducible or repressible (≥1.5 log2-fold) genes after 6 hours stimulation of LPS (LPS-stimulated BMDMs), non-stimulated BMDMs treated with ABX464 (NS-BMDMs ABX464) or LPS-stimulated BMDMs treated with ABX464 (LPS-BMDMs ABX464). (d) Identification of genes regulated by ABX464 in LPS-stimulated BMDMs. RNA samples of three group of cells taken at time point 12 h were analyzed by RNAseq and each group represents the relative expression between ABX464 and control (DMSO) treated samples. Only those genes are represented that were differentially expressed in each group. (e) Validation of elevated transcript levels of IL-22 in ABX464-treated LPS-stimulated BMDMs by qPCR.
