Fig. 5: Ornithine, imidazole and atropine affect Mtb growth.

AMs were infected with Mtb H37Rv. At 2 h post-infection, ornithine and imidazole alone or in combination were added alongside atropine. Intracellular CFUs were demonstrated after 5 days post-infection. a Ornithine and imidazole alone, b in combination of both, c atropine. p < 0.0181, p < 0.0112. n = 5 experiments each. d A representative micrograph of LC3-B, ATG-7, ATG-5 and Beclin-1 in AMs and KCs. Blue: DAPI; green: LC-3B and ATG-7; red: ATG-5 and Beclin-1. Images of multiple fields at 63× magnification were taken with oil immersion. e The number of LC-3B puncta relative to the cell area (10 cells per condition) was quantified using ImageJ. Data shown are representative of five independent experiments. f Total protein was extracted and western blotting was performed. A representative blot depicting the levels of LC-3 and p62/SQSTM1 in the control and Mtb-infected macrophages followed by treatment with ornithine and imidazole is shown. GAPDH was used as a loading control. g Normalized fold intensities of LC-3B and p62 levels in Mtb-infected and ornithine or imidazole treated macrophages compared with the levels in the uninfected control. p < 0.0072, p < 0.019, ns not significant. n = 3 experiments. h Alveolar macrophages were labeled with an autophagy sensor expressing RFP-GFP-LC3B for 48 h. Macrophages were then infected with Mtb followed by treatment with ornithine or imidazole for 72 h. Cells were fixed, and LC-3 was analyzed by confocal microscopy. Scale bars: 5 µm. i Quantification of the number of LC-3B positive autophagosomes (yellow fluorescence) and autolysosomes (red fluorescence) in control and Mtb-infected macrophages treated with ornithine and imidazole. p < 0.0041, p < 0.0034. n = 3 experiments. j AMs from C57BL/6 mice were isolated and transfected with the siRNAs targeting LC-3B, ATG-5, ATG-7 and Beclin-1 or with control siRNA, infected with Mtb and then treated with 10 mM ornithine. After 5 days, CFUs were counted. p < 0.0097, p < 0.0067, p < 0.046, p < 0.0327. n = 5 experiments. Data are presented as the mean ± SD using paired two-tailed t test.