RAW 264.7 cells transform to a well-spread morphology before RANKL-induced cell–cell fusion. (A) Experimental timeline for time-lapse imaging. The frame rate is 30 min per frame. (B) Typical phase contrast image sequences of the cells without RANKL treatment (upper); RANKL-treated, non-fused (middle); and RANKL-treated, fused cells (lower). Arrowheads indicate the analyzed cells in the image. Magenta and green indicate mononuclear cells, and blue indicates a binuclear cell. The numbers in each image indicate frame numbers of sequential images taken every 30 min. Note that cell–cell fusion occurred at frame #15 of RANKL-treated, fused cells (lower). Scale bar: 50 μm. (C and D) The area size (C) and circularity (D) of each cell in B. The color of each line corresponds to each cell. The dotted line indicates the frame in which cell–cell fusion occurred. (E and F) Time-course changes of area size (E) and circularity (F) without RANKL treatment (black); RANKL-treated, non-fused (green); and fused cells (magenta). Time 0 was defined as the frame that cell–cell fusion occurred for RANKL-treated fused cells and an arbitrary time for non–RANKL-treated/RANKL-treated non-fused cells. Mean ± SEM, without RANKL: n = 27 cells, non-fused: n = 30 cells, and fused: n = 28 cells. (G and H) Scatter plots of area size (G) and circularity (H) at time −0.5 h, just before cell–cell fusion. Mean ± SEM, without RANKL: n = 27 cells, non-fused: n = 30 cells, and fused: n = 28 cells. P value obtained from one-way ANOVA with Tukey’s test. *P < 0.05; ***P < 0.001.