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Fig. 2. Knockdown of RhoG suppresses cell spreading. (A) Parental, control RNAi and RhoG RNAi HeLa cells were plated on fibronectin-coated coverslips for 15 minutes, and cells were examined by phase contrast (left panels), or fixed and stained with Alexa Fluor 594-conjugated phalloidin to visualize F-actin (right panels). Bars, 50 µm (left) and 20 µm (right). (B) Quantitative analyses of cell spreading. Cells were plated on fibronectin-coated coverslips for 15 minutes. Average cell area was calculated from the phase contrast images of nine randomly selected fields (top). The number of cells that had fully spread (cell area >1000 µm²) was expressed as a percentage of the total number of cells (bottom). About 100 cells were assessed in one experiment, and data are the means ± s.e.m. of three independent experiments. (C) Control RNAi and RhoG RNAi HeLa cells were plated onto fibronectin-coated dishes, and they were examined by phase contrast at 15, 30 and 60 minutes after plating. Bar, 20 µm. (D) Cell lysates from HEK 293T cells transiently transfected with the indicated plasmids were analyzed by immunoblotting with anti-GFP antibody: GFP-hRhoG, GFP-tagged human RhoG; GFP-mRhoG, GFP-tagged mouse RhoG. (E) RhoG RNAi HeLa cells transiently transfected with GFP or GFP-tagged mouse RhoG (GFP-mRhoG) were plated on fibronectin-coated coverslips. At 15 minutes after plating, they were fixed and stained with Alexa Fluor 594-conjugated phalloidin to visualize F-actin (left panels). The transfected cells were shown by the fluorescence of GFP (right panels). Bar, 20 µm.

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