Fig. 2: Expansion of the lysosomal compartment in RAW 264.7 MΦ in response to treatment with mycobacterial lipid (100 μg/ml for 48 h) visualized with LysoTracker™-Red staining. | Nature Communications

Fig. 2: Expansion of the lysosomal compartment in RAW 264.7 MΦ in response to treatment with mycobacterial lipid (100 μg/ml for 48 h) visualized with LysoTracker™-Red staining.

From: Inhibition of the Niemann-Pick C1 protein is a conserved feature of multiple strains of pathogenic mycobacteria

Fig. 2

a Representative confocal microscopy images of RAW 264.7 MΦ either untreated, DMSO or U18666A treated or incubated with lipid extracts of Mtb clinical strains 281, 333, 639 or H37Rv or M. canettii stained with LysoTracker™-Red. The right image of each pair is a higher magnification of the region indicated in the left image. Nucleus is stained with Hoechst 33342 (blue). Scale bar represents 10 μm. Exposure times for all images was equivalent, except for DMSO indicated as a longer exposure. b Quantification of lysosome frequency per cell in vehicle or U18666A (left histogram) or DMSO and Mtb lipid or M. canettii lipid treated cells (right histogram). Data are means ± SEM, N = 4 replicates per sample comprising a minimum of 50 cells per random field. Statistical analysis, unpaired t test with Welch’s correction for DMSO and U18666A, two-tailed; 1-way ANOVA with Dunnett test, F = 1.945 for DMSO or lipid treated. ****p < 0.0001, other p values as indicated. Data are representative of two independent experiments. c Box-and-whisker plot of lysosome diameters (μm) and statistical distribution of lysosome diameters in vehicle or U18666A treated RAW 264.7 MΦ. Left histogram, data are means ± SEM, N = minimum of 100 lysosomes within four random fields. DMSO: min: 0.149 μm, max: 0.709 μm, center: 0.377 μm; U18666A: min: 0.216 μm, max:1.791 μm, center: 0.793 μm. Box contains 90% of all events. Statistical analysis, unpaired t test with Welch’s correction, two-tailed. ****p < 0.0001. Statistical distribution values indicated are median diameter (μm). d Box-and-whisker plot of lysosome diameter (μm) and statistical distribution of lysosome diameters in DMSO or lipid treated RAW 264.7 MΦ. Data are means ± SEM, N = minimum of 100 lysosomes within four random fields. DMSO: min: 0.149 μm, max: 0.709 μm, center: 0.377 μm; 281: min: 0.081 μm, max: 0.941 μm, center: 0.470 μm; 333: min: 0.204 μμm, max: 0.975 μm, center: 0.456 μm; 639: min: 0.161 μm, max: 1.124 μm, center: 0.436 μm; H37Rv: min: 0.180 μm, max: 1.052 μm, center: 0.456 μm; M. canettii: min: 0.149 μm, max: 1.047 μm, center: 0.383 μm. Box contains 90% of all events. Statistical analysis, 1-way ANOVA with Kruskal–Wallis test, Kruskal–Wallis statistics: 79.61. ****p < 0.0001, other p values as indicated. Statistical distribution values indicated are median diameter (μm). Data are representative of two independent experiments. Source data are provided as a Source data file.

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