Protease inhibition activates the nuclear piston and slows cell velocity. (A) HT1080/MT1 cells were transfected with GFP-MLC2 and RFP-NLS to highlight the trailing edge (TE) and nucleus (N), respectively. Bar, 5 µm. (B) Kymographs of live-cell imaging sequences showing that in control cells, the nucleus (V_N, velocity nucleus) moves synchronously with the trailing edge (V_TE, velocity trailing edge; left panels), whereas in protease inhibitor–treated cells, the nucleus can undergo acceleration away from the trailing edge (right panels). Bars, 5 µM. (C) Examples of instantaneous velocity plots of the nucleus (red) versus the trailing edge (green) showing the independent movement of the nucleus in response to protease inhibition (inh.; bottom) compared with a control cell (top). (D) Pearson’s correlation coefficients between the velocity of the nucleus and trailing edge (n = 12, N = 3). *, P < 0.05 versus control cells. (E) The velocity of HT1080/MT1 tumor cells is reduced upon protease inhibition and activation of the nuclear piston (n = 45, N = 3). Error bars indicate SEM. *, P < 0.01 versus control cells.