Screening of focused small molecule library for inhibitors of MNGC formation. RAW264.7 macrophages were pretreated for 2 h with a collection of 43 compounds active against enzymes involved in epigenetics regulation at a concentration of 20 μM and then infected with 30 MOI of Bp K96243 for 8 h. Cells were fixed, stained in IF and imaged as described above. The effect of the tested compounds on MNGC formation was quantified. Compounds were ranked based on the potency of MNGC inhibition when compared to DMSO-treated, Bp K96243-infected samples (Negative control). Cytotoxic (Number of Nuclei Z-score < -3) were not further considered. (A) Representative confocal images of macrophages pre-treated with DMSO control or primary hit compounds active in the MNGC screen. Scale bar: 90 μm. (B) Compounds that significantly reduced the number of MNGC when compared to DMSO treated samples (% MNGC Z-score > 3) were scored as positive hits (red bars). Bars represent means from two replicates. (C) Dose-dependent inhibition of MNGC formation by compound M-344 identified in the primary screen.