FIG 2

In vivo validation of tamoxifen as an HDT. (A) Bacterial burden assay of mWasabi-expressing Mm-infected zebrafish larvae treated with increasing doses of tamoxifen (2.5, 5, and 10 μM) or control (DMSO at 0.1% [vol/vol]). Treatment was started at 1 hpi, and larvae were anesthetized at 4 dpi for imaging. Representative stereo fluorescent images of whole larvae infected with mWasabi-expressing Mm are shown. Magenta shows Mm; scale bar, 1 mm. (B) Quantification of bacterial burden shown in A. Bacterial burden was normalized to the mean of the control group (set at 100% and indicated with the dotted line). Data from 4 experimental repeats were combined (n = 132 to 139 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 Kruskal-Wallis test with Dunn’s multiple-comparison test. (C) Bacterial burden assay of mWasabi-expressing Mm-infected zebrafish larvae treated with 5 and 10 μM tamoxifen or control (DMSO at 0.1% [vol/vol]). Treatment was started at 1 hpi, and larvae were anesthetized at 1, 2, 3, and 4 dpi for imaging. Bacterial burden was normalized to the control (DMSO at 1 dpi), and data of two experimental repeats were combined (n = 65 to 70 per group). All larvae in the 10 μM group died between 3 and 4 dpi. Box plots with 95% confidence intervals are shown. The black line in the box plots indicates the group median, while the dotted line indicates the group mean. Statistical analysis was performed between treatment groups per time point using a Kruskal-Wallis test with Dunn’s multiple-comparison test. (D) Mm growth in liquid culture during treatment with 5 or 10 μM tamoxifen or control (DMSO at equal volume) up to the assay endpoint, day 2. Lines depict mean ± standard deviation of two experiments. Statistical significance of treatment versus control treatment was tested using a two-way ANOVA with Dunnett’s multiple-comparison test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.