Rotational speed of two-cell colonies of keratinocytes is positively correlated with their proliferative capacity. (A, left) Motion of paired human epidermal keratinocytes. (right) Clonal growth of two-cell colonies was traced and proliferative capacity was evaluated by counting cell number in the colony after 6 d of cultivation with Hoechst 33325. Bars, 20 µm. (B) Correlation between rotational speed in the two-cell colony stage (D1) and proliferative capacity (number of cell doublings) after 6 d of cultivation. Two-cell colonies were categorized in four groups depending on rotational speed: rotation score 1 (<0.125 π/h), rotation score 2 (≥0.125 π/h and <0.25 π/h), rotation score 3 (≥0.25 π/h and <0.5 π/h), and rotation score 4 (≥0.5 π/h). The data shown (n = 126 colonies) were obtained from nine independent experiments. P-value was calculated by Mann-Whitney nonparametric U test. Full set of data is shown in Fig. S1 A. (C) Rotational motion and expression of IVL in two-cell colonies of keratinocytes. IVL expression was categorized in three groups depending on the number of IVL-expressing cells: IVL score 0, no cells; IVL score 1, single cell; and IVL score 2, two cells. Bar, 20 µm. (D) Frequency of two-cell colonies of keratinocytes with each rotation and IVL scores. The data shown (n = 63 colonies) were obtained from triplicate experiments. P-value was calculated by Mann-Whitney nonparametric U test.