FIG 5

Tamoxifen treatment alters lysosomal function and increases mycobacterial lysosomal localization in vitro and in vivo. (A) Confocal microscopy of DsRed-expressing H37Rv-Mtb-infected Mφ2 macrophages treated with 10 μM tamoxifen or DMSO at equal volume for 4 h. Thirty minutes before the experimental endpoint, cells were incubated with LysoTracker Deep Red to stain for acidic 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 acidic vesicles; scale bar, 5 μm. (B) Quantification of LysoTracker signal in A. Lysotracker-positive area (left) and Mtb colocalization with Lysotracker-positive vesicles (right) are shown. 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 paired t test; a.u., arbitrary units. (C) Confocal microscopy of DsRed-expressing Mtb-infected Mφ2 macrophages treated with 10 μM tamoxifen or DMSO at equal volume for 4 h. Cells were fixed at the experimental endpoint, permeabilized using 0.1% Triton-X, stained for TFEB, and counterstained for the nucleus and F-actin using Hoechst 33342 and phalloidin, respectively; scale bar, 5 mm. (D) Quantification of nuclear TFEB intensity in C. Each dot displays the log₂ (FC) of median nuclear TFEB intensity per donor normalized to DMSO (5 donors in total); 95% confidence intervals are indicated. Statistical significance was tested using a paired t test. (E) CFU assay of Mφ2 macrophages infected with H37Rv-Mtb and treated for 24 h with 10 μM tamoxifen, 10 μM tamoxifen in combination with 10 nM bafilomycin (BAF), or control (DMSO at equal volume). Each dot represents a single donor (6 donors in total) and depicts the mean of 3 replicates. Statistical significance was tested using an RM one-way ANOVA with Holm-Sidak multiple-testing correction. (F) Confocal microscopy maximum projection of the indicated ROI in zebrafish larvae treated with 5 μM tamoxifen or control (DMSO at equal volume). Treatment was started at 31 hpf, and at 3 dpf, larvae were immersed in 5 μM LysoTracker Red DND-99 for 1 h and subsequently anesthetized for imaging. Cyan shows acidic vesicles; scale bar, 10 μm. (G) Confocal microscopy maximum projection of mWasabi-expressing Mm-infected 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 immersed in 5 μM LysoTracker Red DND-99 for 1 h and subsequently anesthetized for imaging. Representative maximum projection images of LysoTracker-positive Mm clusters in the CHT region are shown. Cyan shows acidic vesicles, and magenta shows Mm; scale bar, 50 μm. (H and I) Enlargement of areas indicated in G. Cyan shows acidic vesicles, and magenta shows Mm. Arrowheads indicate LysoTracker-positive Mm clusters; scale bar, 10 μm. (J) Quantification of LysoTracker-positive Mm clusters normalized to the control. Data from 3 experimental repeats were combined (n = 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; *, P < 0.05.