Fig. 2: Tubeimosides inhibit NPC1.

A Docking of indicated compounds to NPC1-C and EBOV-GP complex (PDB: 5F1B) was analyzed via Webina. B SNB-19 cells were treated overnight with indicated compounds. After being fixed, cells were stained with filipin (50 µg/mL) and visualized by confocal microscopy (scale bar, 40 μm). Experiments were repeated 3 times independently, and representative results are shown. C Vero-E6 cells were treated with Tubs I/II/III at 0.5 μM and infected with HIV-1 Luc-pseudovirus expressing MARV-GP. Viral infection was determined as previously. D Vero-E6 cells were treated with indicated compounds and infected with HIV-1 Luc-pseudovirus expressing EBOV-GP. Tor, Tam, and Tubs I/II/III were used at 2.5 μM, 5.0 μM, or 0.5 μM, respectively. DMSO was used as a control (Ctr). E Vero-E6 cells were pretreated for 1 h with EIPA and Tubs I/II/III and spinoculated with GFP-labeled EBOV-VLPs expressing EBOV-GP or VSV-G. After removal of VLPs and culture for another 3 h, GFP-positive cells were quantified by flow cytometry. EIPA [5-(N-ethyl-N-isopropyl) amiloride] and Tubs I/II/III were used at 25 μM, or 0.5 μM, respectively. F CTS-B or CTS-L substrates were incubated with cell lysate from A549 cells treated with Tubs I/II/III, and their activity was determined. E-64-d ethyl ester (EST) was used at 10 μM and Tubs I/II/III were used at indicated concentrations. G A549 cells were treated with NH₄Cl at 100 µM and Tubs I/II/III at 0.5 μM. After being stained with LysoTracker Red, cells were visualized by fluorescence microscope (EVOS FL Auto Imaging System) (scale bar, 200 μm). Experiments were repeated 3 times independently, and representative results are shown. Error bars in C and D indicate SEMs (n = 3 biologically independent experiments). One-way ANOVA was applied. ***P < 0.001, ****P < 0.0001; n.s. not significant.