DNT associated with the FN network diffuses from the cell surface and affects sensitive cells. (A) The concentration of DNT diffused from FN-null cells supplemented with hFN (open triangles) or not (closed triangles). The culture supernatant of the cells was obtained as described in Methods, and the DNT concentration was determined. As a control, the medium incubated without FN-null cells (closed squares) was prepared in the same manner. The abscissa indicates the time after the washing of DNT-treated cells. Each plot represents the mean ± S.D. (n = 3). Asterisks indicate significant differences (P < 0.001). (B) Stress fiber-inducing activity of DNT liberated into the culture supernatant. The culture supernatant was obtained 15 min after washing in the experiments of panel A, and examined for DNT activity on MC3T3-E1 cells. Actin fibers were visualized by rhodamine-phalloidin. The left panels show MC3T3-E1 cells incubated with each culture supernatant and the right panels show the cells incubated with DNT. The experiments were performed three times and representative results are shown. Bar, 5 μm.