Figure 1.

Treatment with U18666A or itraconazole increases endolysosomal cholesterol storage in bat-derived cells. (A) Cellular cholesterol pools in MoKi control cells (DMSO-treated) or MoKi cells treated for 16 h with itraconazole (Itra, 2 µg/mL) or the NPC1 inhibitor U18666A (2 µg/mL). Endolysosomes were stained with the organelle-specific marker lysotracker and unesterified cholesterol was visualized using filipin. Heatmaps were generated by colour-encoding filipin-positive pixels according to their intensity values. Representative 2D maximum intensity projections of entire z-stacks obtained by confocal imaging of 3 individual experiments are shown. Scale bar, 20 µm. (B) Overlaps of filipin/LysoTracker signals within the stacks were analyzed by calculating Manders’ coefficients. Bar graphs represent means ± SEM of 9 stacks, with 0 indicating no overlap, and 1 indicating perfect overlap. (C) Global cellular cholesterol levels. Data are expressed as mean cholesterol concentrations (µg/mL) ± SEM from five independent experiments. Statistically significant differences were assessed by one-way ANOVA followed by Dunnett’s multiple comparison test. p-values ≤ 0.05 were considered statistically significant. ****p ≤ 0.0001.