IZUMO1 induces fusion of BHK cells. (A) Representative images of mixed cells transfected with pCI::GFPnes or pCI::H2B-RFP empty vectors or containing the coding sequence for the expression of GCS1/HAP2, IZUMO1, and JUNO as indicated. Arrows show content mixed cells containing both GFPnes (green cytoplasm) and H2B-RFP (magenta nuclei). DAPI staining is shown in blue. Scale bars, 20 µm. See also Fig. S2 A. (B) Quantification of content mixing experiments. The percentage of mixing was defined as the ratio between the nuclei in mixed cells (NuM) and the total number of nuclei in mixed cells and fluorescent cells in contact that did not fuse (NuC), as follows: % of mixing = (NuM/[NuM + NuC]) × 100. Bar chart showing individual experiment values (each corresponding to 1,000 nuclei) and means ± SEM of four independent experiments. Comparisons by one-way ANOVA followed by Dunett’s test against the empty vectors. **P < 0.01, ****P < 0.0001. (C) Scheme of experimental design. (D) Representative images of cells transfected with empty or IZUMO1-coding vectors after a content mixing assay and subjected to immunostaining using an anti-V5 antibody. Each separate channel (red, green, far red, and DAPI) and merge are shown. Scale bars, 20 µm.