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. Author manuscript; available in PMC: 2010 Nov 1.
Published in final edited form as: Nat Cell Biol. 2010 Apr 18;12(5):477–483. doi: 10.1038/ncb2049

Figure 2. RhoGDI1 depletion impairs cell migration.

Figure 2

(a) Time-course analysis of closure of a wound generated on a confluent monolayer of control or RhoGDI1 siRNA transfected HeLa cells. The graph depicts the wound area with error bars representing standard deviation (n=10). (b) Time-course analysis of closure of a wound generated on a confluent monolayer of control or RhoGDI1 siRNA transfected WM2664 melanoma cells. The graph depicts the wound area with error bars representing standard deviation (n=6). (c) Analysis of the velocity of control or RhoGDI1 siRNA transfected WM2664 melanoma cells. Data were analyzed by two-tailed unpaired student’s t test (p= 1.72×10−14) with error bars representing standard error of the mean (s.e.m.) from respectively n=28 and n=56 cells. The average speed of control and RhoGDI1 knock-down cells was 12.74 µm/min and 5.64 µm/min respectively. (d) WM2664 melanoma cells were transfected with control or RhoGDI1 siRNA for 72h. Active Rac1 and active RhoA were pulled-down from cell lysates with GST-PBD or GST-RBD beads respectively. Bound proteins and total cell lysates were resolved by SDS-PAGE and analyzed by Western blotting. (e) Cell lysates of control or RhoGDI1 siRNA transfected HeLa cells were resolved by SDS-PAGE and analyzed by Western blotting. Notice that Rho protein effectors are not activated despite the strong activation of RhoA and Rac. (f) Images extracted from supplementary information movie 1 depicting one WM2664 melanoma cell transfected with fluorescently labeled RhoGDI1 siRNA (red arrowhead) and two WM2664 cells transfected with unlabelled control siRNA (white and black arrowheads) migrating over time. Panel on right shows an image taken at t0 used to identify the cell transfected with the fluorescently labelled siRNA. Scale bar is 40 µm. (g) Representative XY migration tracks of control (left) or RhoGDI1 (right) siRNA transfected WM2664 melanoma cells. The positions of the cells were recorded every 5 minutes over a period of 2 hours (n=10). (h) HeLa cells transfected with control or RhoGDI1 siRNA for 72 h were fractionated into cytosolic or membrane fractions. Membrane fractions were further separated into plasma membrane (PM) and ER membrane (ER) by centrifugation on an iodixanol density gradient. Each fraction was analyzed by SDS-PAGE and Western blotting. (i) Densitometric analysis of the relative intensity of RhoA, Rac1 and Cdc42 bands in each fraction. Notice that upon RhoGDI1 silencing, the GTPases essentially disappear from the plasma membrane fractions.