RhoC is essential for protrusion formation and directional protrusion during chemotaxis. (A) Still images from time-lapse movies of control- and RhoC-siRNA-treated MTLn3 cells stimulated with EGF. Dotted lines show the cell edge. (B) Quantification of protrusive activity in response to EGF in control- and RhoC-siRNA-treated cells. Membrane protrusion is standardized to time 0. Number of cells analyzed: control siRNA = 26 cells, RhoC siRNA = 28. (C) Still images from time-lapse movies of control- and RhoC-siRNA-treated MTLn3 cells stimulated with an EGF-filled micropipette (positioned at the asterisk). The white arrows indicate the resulting directions of protrusion. (D) Standardized membrane protrusion at the front and the back of control- and RhoC-siRNA-treated MTLn3 cells during the 10 minutes after EGF stimulation. Number of cells analyzed: control siRNA = 23 cells, RhoC siRNA = 18 cells. (E) The average chemotactic index of control- and RhoC-siRNA-treated MTLn3 cells over 10 minutes expressed as the cosine (θ). Number of cells analyzed: control siRNA = 24 cells, RhoC siRNA = 11 cells. (F) The polarity index, which corresponds to the angle at which the maximum protrusion is occurring in reference to the pipette position at each time point. The cosine values of these angles are calculated, and these values represent the polarity index of the cell at each time point after stimulation (see Materials and Methods for a detailed description). The polarity indices from different cells are averaged and then plotted versus time after stimulation. Number of cells analyzed: control siRNA = 20 cells, RhoC siRNA = 24 cells. Scale bars: 10 µm. *P<0.05. Error bars indicate the s.e.m.