Content mixing assays of BHK and HEK293T cells. (A) Evaluation of content mixing of BHK cells. Images from Fig. 2 A in each separate channel (red, green, and DAPI) and merge. Scale bars, 20 µm. (B) Evaluation of cell fusion by content mixing of human embryonic kidney HEK293T cells, related to Fig. 2. Images of mixed cells transfected with pCI::GFPnes or pCI::H2B-RFP empty vectors or containing the coding sequence for the expression of GCS1, IZUMO1, and JUNO as indicated in each panel. Arrows show fused cells containing green cytoplasm (GFPnes) with red nuclei (H2B-RFP). DAPI staining, blue. Each channel (red, green, and DAPI) and merge are shown. Scale bars, 20 µm. (C) Quantification of content mixing of HEK293T cells. The percentage of mixing is 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 content mixing = (NuM/[NuM + NuC]) × 100. Graph shows experiment values and means ± SEM of four independent experiments. Comparisons by one-way ANOVA followed by Dunnett’s test against the empty vectors. **P < 0.01, ***P < 0.001.