Amiodarone controls Mav infection in primary human macrophages by promoting antimycobacterial autophagy and activating master autophagy regulator TFEB. (A) Western blot analysis of autophagy markers in M2 macrophages treated for 24 hours with 10 µM amiodarone or an equal volume of vehicle control dimethyl sulfoxide (DMSO) (0.1%, vol/vol) in the presence or absence of bafilomycin A1 (Baf) (10 nM) during Mav infection. Shown are blots from one representative donor out of six donors tested. The image depicts the boxed lanes from the unprocessed original images (Fig. S2). (B) Quantification of LC3-II (+Baf) protein levels from panel A. Protein levels were first normalized to actin and subsequently compared to DMSO control (=100%, indicated with the dotted line) per donor. Data represent the mean ± SD from different donors (n = 6). Statistical significance was tested using a repeated-measures one-way ANOVA with Bonferroni’s multiple comparison correction. (C) Quantification of p62 (+Baf) protein levels from panel A. Protein levels were first normalized to actin and subsequently compared to DMSO control (=100%, indicated with the dotted line) per donor. Data represent the mean ± SD from different donors (n = 5). Statistical significance was tested using a repeated-measures one-way ANOVA with Bonferroni’s multiple comparison correction. (D) M2 macrophages were treated for 24 hours with 10 µM amiodarone or an equal volume of vehicle control DMSO (0.1%, vol/vol) after infection with Wasabi-expressing Mav (green). Cells were subsequently stained with LC3-II (red) and Hoechst 33342 (blue) and analyzed by confocal microscopy. Images shown are of one representative donor out of eight donors tested. Arrows indicate colocalization of Mav-Wasabi with LC3-II puncta. (E) Quantification of the LC3-II area per cell count. Dots represent the mean from three wells (three images/well) per condition of a single donor (n = 8). Data are expressed as the percentage of vehicle control DMSO (=100%, indicated with the dotted line) per donor. Statistical significance was tested using a Wilcoxon matched-pairs test. (F) The percentage colocalization (indicated by white arrows in panel D) of intracellular mycobacteria with LC3-II puncta was determined. Dots represent the mean from three wells (three images/well) per condition of a single donor (n = 8). Statistical significance was tested using a Wilcoxon matched-pairs test. (G) M2 macrophages were treated for 4 hours with 10 µM amiodarone or an equal volume of vehicle control DMSO (0.1%, vol/vol) in the presence or absence of 10 nM Baf after infection with Wasabi-expressing Mav (green). Cells were subsequently stained with p62 (red) and Hoechst 33342 (blue) and analyzed by confocal microscopy. Images shown are of one representative donor out of five donors tested. Arrows indicate colocalization of Mav-Wasabi with p62. (H) Quantification of the p62 area per cell count. Dots represent the mean from three wells (three images/well) per condition of a single donor (n = 5). Data are expressed as the percentage of vehicle control DMSO (=100%, indicated with the dotted line) per donor. Statistical significance was tested using a Wilcoxon matched-pairs test. (I) The percentage colocalization (indicated by white arrows in panel G) of intracellular mycobacteria with p62 was determined. Dots represent the mean from three wells (three images/well) per condition of a single donor (n = 5). Data are expressed as the percentage of vehicle control DMSO. Statistical significance was tested using a Wilcoxon matched-pairs test. (J) Confocal microscopy of Wasabi-expressing (green) Mav-infected M2 macrophages treated with 10 µM amiodarone or an equal volume of vehicle control DMSO for 4 hours. Cells were subsequently stained for TFEB (yellow) and Hoechst 33342 (blue). Shown are images of one representative donor out of seven donors tested. (K) Quantification of the total intensity of TFEB within the mark of the cell nucleus. Data represent the mean ± SD from different donors (n = 7). Dots represent the mean from three wells (three images/well) per condition of a single donor. Data are expressed as the percentage of vehicle control DMSO (=100%, indicated with the dotted line) per donor. Statistical significance was tested using a paired t-test. (L) Bacterial survival of Mav within M2 macrophages after treatment for 24 hours with 10 µM amiodarone or an equal volume of vehicle control DMSO in the absence or presence of 10 nM Baf. Cells were subsequently lysed and bacterial survival was determined by MGIT assay. Data represent the mean ± SD from different donors (n = 6). Dots represent the mean from triplicate wells of a single donor. Bacterial survival is expressed as the percentage of vehicle control DMSO (=100%, indicated with the dotted line) per donor. Statistical significance was tested using a repeated-measures one-way ANOVA with Bonferroni’s multiple comparison correction. ns, non-significant; *P < 0.05; **P < 0.01; and ***P < 0.001.
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