Fig. 3: 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 anti-LAMP-1 staining. | Nature Communications

Fig. 3: 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 anti-LAMP-1 staining.

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

Fig. 3

a Confocal microscopy images of RAW 264.7 MΦ treated with vehicle or U18666A and stained with anti-LAMP-1 antibody (green). b Images of RAW 264.7 MΦ treated with DMSO or Mtb lipids or M. canettii lipid. The right image for each pair is a higher magnification of the region indicated in the left image. Nucleus is stained with DAPI (blue). Scale bar represents 10 μm. c Box-and-whisker plot of LAMP-1 positive compartment diameters (μm) and statistical distribution of LAMP-1 positive compartment 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: 0.051 μm, max: 1.190 μm, center: 0.446 μm; U18666A: min: 0.216 μm, max:4.894 μm, center: 0.964 μm. Box contains 90% of all events. Statistical analysis, Mann-Whitney test, two-tailed, ****p < 0.0001. Indicated statistical distribution, values are median diameter (μm). d Box-and-whisker plot of LAMP-1 positive compartment diameters (μm) and statistical distribution of LAMP-1 positive compartment 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: 0.051 μm, max: 1.190 μm, center: 0.446 μm; 281: min: 0.255 μm, max: 2.243 μm, center: 0.582 μm; 333: min: 0.204 μm, max: 2.000 μm, center: 0.560 μm; 639: min: 0.253 μm, max: 2.247 μm, center: 0.582 μm; H37Rv: min: 0.180 μm, max: 2.166 μm, center: 0.600 μm; M. canettii: min: 0.161 μm, max: 1.232 μm, center: 0.497 μm. Box contains 90% of all events. Statistical analysis, 1-way ANOVA with Kruskal–Wallis test, Kruskal–Wallis statistics: 87.02. ****p < 0.0001. MΦ statistical distribution, values indicated are median diameter (μm). Data are representative of two independent experiments. e Q-PCR of relative LAMP-1 transcript expression in RAW 264.7 MΦ, normalized to β-actin. Data are mean ± SEM, N = 5 replicates per sample. Statistical analysis, t test with Welch’s correction, two tailed; and Mann–Whitney test, two-tailed. significance value as indicated. f Left, Western blot of LAMP-1 and β-actin protein expression in untreated, vehicle, U18666A and H37Rv lipid treated RAW 264.7 MΦ. g Quantification of LAMP-1 protein expression relative to β-actin Data are mean ± SEM, N = 2 replicates per sample. Statistical analysis, t test with Welch’s correction No statistically significant differences, two-tailed. Data shown are representative of two independent experiments. Source data are provided as a Source data file.

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