Figure 3.

Pharmacological inhibition of triglyceride targeting lipases prevents the initiation of LD modulation early in R. conorii THP-1 macrophage infection. THP-1 macrophages were treated with Orlistat (10 μM) 24 hr prior to infection with R. conorii (MOI of 2). Samples for LD analysis were collected at 1 hour post infection (hpi) before being stained with Oil Red O for visualization of LDs. Ten fields of view from three independent experiements with 3–15 cells per field were quantified to define (A) average lipid droplets (LDs) per THP-1 cell and (B) average area (μm²) for all groups using ImageJ software with a constant threshold. (C) A representative visualization at 100X of one cell within each treatment group is shown. Oil red O (red) signifies LDs, α-Rickettsia (green) signifies R. conorii, and DAPI (blue) signifies nuclei. White bar is indicative of 10μm. (D) Rickettsial survival in the presence of Orlistat or DMSO was quantified by qPCR to analyze R. conorii (sca1) per host cell (actin) at 1 day post infection (dpi), 3 dpi, and 5 dpi. Data is representative of three independent experiments with each condition performed in triplicate. Significance is represented by p≤0.05 determined by a one-way ANOVA followed by Bonferroni’s correction post hoc test. Statistical significance is defined by *p≤0.05, **p≤0.005, ***p≤0.001.