X applications suite software

Statistical analyses were conducted using GraphPad Prism 9. Error bars in the figures represent means with standard error. In Figs 1b and 1c, Figs. 3b and 3d, Fig. 4b, and Fig. S2b, comparisons between viruses were made using a one-way ANOVA with Bonferroni post-test. Neutralization titers were determined using least-squares non-linear regression. In Figs 2a–c, error bars represent geometric means with 95% confidence intervals. Comparisons between viruses in these figures were made using a repeated measures one-way ANOVA with Bonferroni post-test. To better approximate normality, comparisons were conducted using log10 transformed NT₅₀ values. Error bars in Fig. 2d represent means ± standard deviation. Cell-cell fusion and syncytia formation shown in Figs. 3a, 3c, and Fig. S2a was quantified using the Leica X Applications Suite software. Spike processing shown in Fig. 4c was quantified by NIH ImageJ; the values are then set relative to D614G, with D614G = 1.0.

Direct spike-mediated cell to cell fusion assays were performed by first co-transfecting 293T cells with spike and GFP. 293T cells were incubated 24 hours then detached and reseeded in a plate containing one of two detached target cells; 293T-ACE2 or CaLu-3. 293T-ACE2 cells were incubated for 6.5 hours and CaLu-3 cells 4 hours then fusion was imaged using a Leica Dmi8 microscope. Areas of fusion were quantified using the Leica X Applications Suite software to outline the edges of fields of GFP and quantify then areas. Three images from duplicate wells were randomly taken. Scale bars represent 150 μM and one representative image was selected for presentation.