Fig. 3. Syncytium formation is not driven by membrane tension and requires metabolic energy. (A) To explore the effects of a decrease in membrane tension on the extent of syncytium formation (black bars) and the mean radius of the cells (gray bars), immediately after a 1-minute application of acidic-pH buffer we placed Sf9^Op1D cells into either PBS (control) or PBS supplemented with 250 mM stachyose (hypertonic medium). The syncytium-formation and cell-radii analyses were carried out 30 minutes after low-pH application for >1330 and >22 cells, respectively. The syncytium index and mean cell radius in hypertonic medium were normalized to those in the control experiments. (B,C) The metabolic inhibitor sodium azide (NaN₃, 5 mM) did not prevent early fusion stages but blocked syncytium formation. (B) An overlay of a representative field of red and green fluorescence of the NaN₃-treated cells fixed 1-hour after a 1-minute low-pH pulse. Membrane merger between cells that were pre-labeled with the red membrane dye Vybrant DiI and cells pre-labeled with content probe CellTracker Green yielded double-labeled cells. Unfused cells are seen as only red or only green ones. (C) Local fusion between cells that were pre-labeled with different probes in the presence of NaN₃ and between controls was quantified as an average number of nuclei in double-labeled cells per field of view. Syncytium formation after a 1-minute low-pH pulse was quantified as the percentage of nuclei in syncytia. The total number of analyzed nuclei for each condition was >2500. A and C show means ± s.d. of ten replicates in a representative experiment, performed twice.