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FIG 4
Tamoxifen treatment modulates autophagy in infected human macrophages and zebrafish. (A) Confocal microscopy of DsRed-expressing H37Rv-Mtb-infected Mφ2 macrophages treated with 10 μM tamoxifen or control (DMSO at equal volume) for 4 h. Thirty minutes before the experimental endpoint, cells were incubated with Cyto-ID to stain for autophagy-related vesicles, fixed with 1% paraformaldehyde, and counterstained for the nucleus using Hoechst 33342. In the representative images, yellow shows the nucleus, magenta shows Mtb, and cyan shows autophagy-related vesicles; scale bar, 5 μm. (B) Quantification of Cyto-ID signals in A. Cyto-ID-positive area (left) and Mtb colocalization with Cyto-ID-positive vesicles (right) are displayed. Each dot displays the mean of 3 or 4 replicates and represents a single donor (4 donors in total), with the median indicated by colored bars. Statistical significance was tested using a Wilcoxon matched-pairs signed-rank test; a.u., arbitrary units. (C) Confocal microscopy of transgenic GFP-Lc3 zebrafish larvae treated with 5 μM tamoxifen or control (DMSO at equal volume). Treatment was started at 3 dpf, and larvae were fixed with 4% paraformaldehyde at 4 dpf for imaging. Representative max projection images of GFP-Lc3-positive vesicles in the indicated region of imaging (ROI) in the tail fin are shown. Cyan shows GFP-Lc3-positive vesicles; scale bar, 10 μm. (D) Quantification of GFP-Lc3 structures shown in C. Data were normalized to the control, and data from 2 experimental repeats were combined (n = 16 to 18 per group). Each dot represents a single larva. Box plots with 95% confidence intervals are shown. The black line in the box plots indicates the group median, while the black line in the dot plot indicates the group mean. Statistical analysis was performed using a Mann-Whitney test. (E) Confocal microscopy of mCherry-expressing Mm-infected transgenic GFP-Lc3 zebrafish larvae treated with 5 μM tamoxifen or control (DMSO at equal volume). Treatment was started at 1 hpi, and at 2 dpi, larvae were fixed with 4% paraformaldehyde for imaging. Representative max projection images of the ROI in the caudal hematopoietic tissue (CHT) region are shown. Cyan shows GFP-Lc3-positive vesicles, and magenta shows Mm; scale bar, 50 μm. (F and G) Enlargement of areas indicated in E. Cyan shows GFP-Lc3-positive vesicles, and magenta shows Mm. Arrowheads indicate GFP-Lc3-positive Mm clusters; scale bar, 10 μm. (H) Quantification of GFP-Lc3-positive Mm clusters in the CHT region shown in E normalized to the control (n = 8 per group). Each dot represents a single larva. Box plots with 95% confidence intervals are shown. The black line in the box plots indicates the group median, while the black line in the dot plot indicates the group mean. Statistical analysis was performed using a Mann-Whitney test; ****, P < 0.0001; ns, not significant.