Fig. 3.

Activity of TBA161 variants in macrophage and zebrafish infection models. (A) THP-1 macrophages were infected with Mtb carrying pTetDuo, expressing GFP with the tetracycline-inducible promoter and tdTomato under the constitutive promoter p_smyc. Infected macrophages were treated with various doses of each test compound for 6 days. GFP expression was induced by the addition of ATc, and macrophage nuclei were stained with Hoechst dye to detect macrophages (grey bars). The GFP signal within each macrophage was quantified, representing the amount of viable bacteria (green bars). DMSO- and rifampicin (RIF, 3 µM)-treated samples served as a negative and positive control, respectively. Data points represent the average of duplicates with s.d. (B) Representative images of Mtb-pTetDuo infected THP-1 macrophages treated with DMSO or compound TBA161-C at 6 dpt. Blue, macrophage nuclei (Hoechst); yellow, merged signal of Mtb expressing tdTomato (red) and gfp (green). Scale bars: 50 µM. (C) Dose-dependent activity of TBA161 variants in the zebrafish-M.marinum infection model. Each data point represents the integrated red fluorescence intensity of a single zebrafish embryo, and the signal of each group (10-20 embryos) is expressed as mean±s.e.m. Statistical significance was determined by one-way ANOVA, following Dunnett's multiple comparison test by comparing the signal from the DMSO-treated control sample with each treatment group (**P?0.01, ***P?0.001, ****P?0.0001). (D) Representative images of M. marinum-tdTomato yolk-infected zebrafish embryos treated with DMSO (left) or TBA161-C (right) at 3 dpt. (E) Survival curves of M. marinum yolk-infected zebrafish embryos after dose-dependent drug treatment. The treatment started at 1 dpi by adding compounds to the fish water. Each treatment group consisted of 25-30 embryos. Scale bars: 1 mm.