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. 2020 Jan 28;10:3051. doi: 10.3389/fmicb.2019.03051

FIGURE 6.

FIGURE 6

The effect of NSC 18725 on autophagic flux and intracellular mycobacterial growth. (A) THP-1 cells were pre-treated with 25 μM NSC 18725 for 12 h and Baf-A1 treatment was performed as described in section “Materials and Methods.” At designated time points, cells were fixed, stained with anti-LC3, and immunofluorescent images were captured using a confocal microscope. The image shown is representative of three independent experiments. Scale bar given is 10 μM. (B) The formation of LC3 puncta in panel (A) in different samples were quantified in a random manner (n = 50). The data shown on y-axis is mean ±SE of LC3 puncta formation per cell obtained from three independent experiments. (C) Quantitative data depicting normalized values of LC3 puncta formation in 18725 treated THP-1 cells in the absence or presence of Baf-A1. (D) MDC staining of macrophages pre-treated with NSC 18725 in the presence or absence of Baf-A1 was performed as described in section “Materials and Methods.” The images shown in this panel are representative of experiments performed in duplicates. Scale bar given is 10 μM. (E) THP-1 cells were infected with GFP labeled M. bovis BCG for 4 h at a MOI of 1:10 before treating with NSC 18725 for 12 h. In few combinations, Baf-A1 was added as described earlier before staining with LC3 antibody followed by visualization under confocal microscope. This panel represents the cumulative quantification depicting co-localization between phagosomes and LC3 in three independent experiments and data is represented as mean ± SE (F) The images shown in this panel are representative of experiments performed in triplicates. Scale bar given is 10 μM. (G,H) THP-1 macrophages were infected with either Mycobacterium smegmatis with MOI of 1:1 (G) or Mycobacterium bovis BCG with MOI 1:10 (H) and anti-tubercular activity of NSC 18725 against intracellular mycobacteria was determined as described in section “Materials and Methods.” (I) The antimycobacterial activity of NSC 18725 against intracellular M. smegmatis and M. bovis BCG was determined in the presence of 3-MA as described in section “Materials and Methods.” The data shown in this panel is mean ± SE of bacterial numbers obtained from three or four independent experiments. P < 0.001, P < 0.01, P < 0.05 are represented as ***, **, and *, respectively.