Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cryptococcosis, tuberculosis, and a kidney cancer fail to fit the atherosclerosis paradigm for foam cell lipid content.

doi: 10.1093/jimmun/vkaf038

Figure Lengend Snippet: Figure 3. Characterization of lipid droplets induced in human primary macrophages by C. neoformans infection and by exposure to conditioned medium from ACHN cell cultures, and comparisons between C. neoformans- and M. tuberculosis-induced effects. Panels (A–D) show data obtained with monocyte-derived macrophages (MDM) infected with C. neoformans for 24 h (MOI ¼ 4). Panel (D) also includes cells infected with M. tuberculosis for 24 h (MOI ¼ 4). Panels (E–H) show data obtained with MDM left untreated and treated with ACHN-conditioned medium for 7 days. In all experiments MDM were characterized as CD11cþ cells, which is expressed at levels comparable to the CD68 marker (Fig. S8). In all bar graphs, each dot corresponds to one human donor. (A) Lipid droplet imaging. Representative images of MDM uninfected (leftmost panel), infected with mCherry-tagged C. neoformans, and stained with Bodipy 493/503 (neutral lipid dye, green fluorescence) were acquired by imaging flow cytometry at 24 h post-infection. The 2 rightmost panels show macrophages in the infected culture wells carrying and not carrying intracellular fungi (orange fluorescence). (B) Lipid droplet content was expressed as median fluorescence intensity (MFI; ± SD) of Bodipy 493/503, as obtained by imaging flow cytometry. (C) Neutral lipid measurements. TAG and cholesterol were measured in uninfected and infected cells, as indicated, using a commercially available kit. The box plots show lower quartile, median, and upper quartile of the distribution of multiple donors. The whiskers represent minimum and maximum values. The plus symbol indicates the mean. ns, non-significant; P < 0.05 (paired t-test). (D) Effect on lipid droplet content of treatment with chemical inhibitors. DMSO (vehicle control), 0.4 nM rapamycin (mTORC1 inhibitor), or 30 nM DGAT-1 inhibitor (DGAT-i) (A922500; PubChem CID: 24768261) were added for the duration of infection. Lipid droplet content was quantified by imaging flow cytometry and expressed as Bodipy MFI, as in panel (A). Results are shown as ratios of Bodipy MFI of drug-treated to vehicle-treated infected cells. Mean and SD are shown. ns, non-significant; P < 0.01 (unpaired t-test). (E) Lipid droplet imaging. Cells were stained with Bodipy 493/503 at the end of treatment and images were acquired by imaging flow cytometry, as in panel A. (F) Lipid droplet content was expressed as MFI of Bodipy 493/503, as in panel B. (G) Neutral lipid measurements. TAG and cholesterol were measured in untreated and ACHN-medium-treated cells and data expressed as described in panel C. (H) Effect on lipid droplet content of treatment with chemical inhibitors. ACHN-medium-treated MDM were treated with DMSO (vehicle control), 90 nM DGAT-1 inhibitor (DGAT-i) (A922500; PubChem CID : 24768261), or 10 µM ACAT inhibitor (ACAT-i) (CAS 615264-52-3; PubChem CID : 10019206) for 7 days. Results are shown as ratios of Bodipy MFI of drug-treated to vehicle-treated cells, as in Panel D.

Article Snippet: The following concentrations were used: 30 to 90 nM DGAT inhibitor A922500 (PubChem CID: 24768261) (Santa Cruz Biotechnology, Dallas, Texas, USA), 0.4 nM rapamycin (mTORC1 inhibitor) (Selleckchem, Houston, Texas, USA), and 10 μM ACAT inhibitor CAS 615264-52-3 (PubChem CID: 10019206) (Santa Cruz Biotechnology, Dallas, Texas, USA).

Techniques: Infection, Derivative Assay, Marker, Imaging, Staining, Fluorescence, Flow Cytometry, Control