Fig. 1

PDIMs contribute to phagosome damage and help avoid a phagolysosomal fate in hLECs, which were infected with Mtb WT, Mtb ΔPDIM or Mtb ΔPDIM::PDIM (all GFP tagged) for 5 h before any non-phagocytosed bacteria were washed away. The infection proceeded for 2, 24, 48 or 72 h before fixation. The samples were subject to immunofluorescence labelling using antibodies against (a) Galectin 8 (Gal8) or (c) Cathepsin D (CtsD). Nuclei were visualised with DAPI. White boxes outline the zoomed area below which the fluorescent channels are displayed separately. Scale bars are 10 μm. Images are representative examples from samples at 5 + 24 hpi. Images such as in (a) and (c) were quantified using ImageJ to measure the association of (b) Gal8 or (d) CtsD to each strain of Mtb at each time point. The percentage of Mtb that was positive for the marker [defined as the marker (the red channel) having a mean pixel intensity of at least 100 in the area overlapping with the individual Mtb particles (the green channel)] in each condition is shown. At least six fields of view were imaged per sample per replicate and three independent replicates were performed. The total number of analysed Mtb particles is also displayed for each condition (N). The overall mean is shown with error bars representing the standard error of the mean. Statistical significance was determined using one-way ANOVA with Tukey’s post-test. CtsD Cathepsin D, Gal8 Galectin 8, GFP green fluorescent protein, hLEC human lymphatic endothelial cell, hpi hours post-infection, Mtb Mycobacterium tuberculosis, PDIM phthiocerol dimycocerosate, WT wild type, *** p < 0.001