(A) p27^Kip1-mediated morphological elongation. SNU449Cp cells were transiently transfected with FLAG-p27^Kip1 plasmid. After 48 hours, cells were triple-stained with DAPI, phalloidin conjugated with rhodamine, and anti-FLAG– and FITC-conjugated antibody, before confocal microscopic analysis. (B) Reversion of elongated SNMU449Tp cells to control cell–like cells via removal of cytosolic p27^Kip1 and concomitant introduction of nuclear p27^Kip1S10A. SNU449Tp cells were infected with siRNAp27^Kip1 adenovirus for 24 hours. Cells were then transfected with FLAG-p27^Kip1S10A mutant and incubated for an additional 24 hours, prior to triple-staining, as in A. Note that mutant p27^Kip1-transfected cells (with a green-positive nucleus) have well-developed stress fibers, compared with adjacent untransfected cells. Two images for the same condition are shown. (C) Regulation of p27^Kip1 levels. Cells, as in B, were immunoblotted. (D) p27^Kip1-dependent RhoA activity. Cells were infected with Ad-control or Ad-sip27^Kip1 for 24 hours, before being harvested for use in RhoA assay. (E) Reversion of morphological changes by active RhoA. SNU449Tp cells were cotransfected with pEGFP and active RhoA 63L. Actin was stained. (F) RhoA-mediated regulation of cytosolic p27^Kip1 and morphology. SNU449Tp cells were cotransfected with pEGFP plus active mDia (mDiaΔ3). (G) Huh7 cells were immunostained for DAP1 and p27^Kip1, 48 hours after cotransfection with pEGFP and mDia1Δ3. Data shown represent 3 independent experiments. Scale bars: 10 μm (E); 20 μm (B, F, and G); 50 μm (A).