Fig 4. Addition of cations (Ca⁺⁺, K⁺), cathepsins (B and L) or an anionic lipid (BMP) does not trigger detectable fusion between cells expressing EBOV-GP_cl and cells expressing NPC1 C-loop.

Target and effector HEK293T/17 cells were prepared, treated and analyzed for CCF as described in the legend to Fig 1A except that the pH pulse was done, as indicated, in fusion buffer containing (A) 2 mM Ca²⁺ or 140 mM K⁺ or (B) a mixture of 5 μg/ml of pre-activated cathepsin B and 5 μg/ml of pre-activated cathepsin L (see S5 Fig for cathepsin activities). (C) Effector and target HEK293T/17 cells were prepared as described in the legend to Fig 1A. Cocultured cells were treated with an equimolar mixture of 5 μM BMP and delipidated BSA for 5 min at 10°C, and then subjected to a 5 min pulse at 37°C at the indicated pH. Results in each panel represent mean +/- SD of triplicate samples from one of two experiments with similar results. Statistical analyses are based on two-way ANOVA tests; samples were compared to respective No GP samples. ****p < 0.0001.