Figure 6.

Cdc42 functions upstream of FBP17 not only in RasV12-transformed cells but also in the neighboring normal cells

(A) Cdc42 activity assay in single or mix cultures of MDCK and MDCK-pTR GFP-RasV12 cells. At 16 hr after the induction of RasV12, cells were treated with GST-WASP-CRIB proteins and then stained with anti-GST antibody. RasV12 cells stably expressing Luciferase (Luc)-shRNA were used as control for RasV12 FBP17-shRNA1 cells.

(B) Quantification of the cdc42 activity. Values of pixel intensity in the cytoplasm were measured and expressed as a ratio relative to RasV12 Luc-shRNA. Differences of pixel intensity between GST-WASP-CRIB and GST were calculated in each condition. Data are mean ± SD. ∗∗p < 0.01 (unpaired two-tailed Student's t-tests).

(C and D) Fluorescence images of RasV12-transformed (C) or normal (D) cells expressing mCherry-FBP17 in the mix culture without or with the cdc42 inhibitor ML141. (D) z stack images are shown. The asterisks indicate RasV12 cells. (A, C, D) Scale bars, 20 μm.

(E) EM images of the interface between normal and RasV12 cells (upper panels) or between RasV12 cells (lower panels) in the mix culture without or with ML141. Tracings of cell membranes were shown in blue or red. The brackets indicate the position of cell-cell contacts. Scale bar, 1 μm.

(F) Waviness of cell membranes at each cell-cell contact in mix culture without or with ML141. As for MDCK-RasV12 boundary, the data on the underlined side is shown. Data are median ±SD. ∗∗p < 0.01 (unpaired two-tailed Student's t-tests).