Surface localization of IZUMO1 and JUNO; induction of multinucleation of BHK cells by IZUMO1, related to Fig. 1 . (A) IZUMO1 presence on the surface of BHK cells transfected with pCI::IZUMO1-V5::H2B-RFP was determined by immunostaining using an anti-IZUMO1 (Mab 120) that recognizes the extracellular N-terminal region of the protein (green) or anti-V5 against the cytoplasmic C-terminal tag (magenta). Non-permeabilized and permeabilized cells were tested. Non-permeabilized cells were exposed to anti-IZUMO1, then permeabilized and finally incubated with anti-V5 (lower row). In all cases, DAPI staining is shown in blue in the merge. Scale bars, 20 µm. (B) JUNO localization to the plasma membrane of BHK transfected with pExpress1::JUNO-flag and pCI::H2B-RFP was evaluated by incubating the cells with anti-flag antibody before fixation (non-permeabilized) or after fixation and permeabilization. The immunostaining signal (green) and the nuclei of transfected cells (magenta) are shown in separate channels and in the merge, which includes the DAPI staining (blue). Scale bars, 20 µm. (C) Images from Fig. 1 D in each separate channel (green in gray, magenta, and DAPI) and merge. The numbers in the merged images correspond to the confocal planes in micrometers. Note that two nuclei in magenta and two in blue suggest the recent fusion between cells expressing IZUMO1::H2B-RFP (magenta) with untransfected cells (blue). See also Video 1. Scale bar, 20 µm. (D) WT or cells with doxycycline (DOX)-inducible expression of IZUMO1-GFP were evaluated for multinucleation in the presence or absence of the inhibitor FdUrd. Different concentrations of DOX were tested. WT and uninduced cells were stained with CellMask Green. The percentage of multinucleation was defined as the ratio between the nuclei in multinucleated cells (NuM) and the total number of nuclei in multinucleated and fluorescent cells in contact that did not fuse (NuC), as follows: % of multinucleration = (NuM/[NuM + NuC]) × 100. We show individual data and means ± SEM of four to six independent experiments. Comparisons were made with two-way ANOVA followed by Bonferroni’s multiple comparisons against the WT (in black) or between FdUrd and control (in red). **P < 0.01, ***P < 0.001, ****P < 0.0001.