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. 2014 Mar 4;110(8):2099–2108. doi: 10.1038/bjc.2014.99

Figure 3.

Figure 3

Rho signalling controls transendothelial migration towards AA. (A) Invasion assays with PC3-GFP cells were performed using cell-culture inserts (8 μm pore size) coated by a synthetic basement membrane (Matrigel) (left panel) or culture inserts (8 μm pores) coated by a layer of BMEC above a synthetic basement membrane (Matrigel) (right panel). 2 × 105 PC3-GFP cells pre-incubated with Y27632 (40 μM) or the vehicle (H2O) for 30 min were added to the top of the inserts and allowed to invade towards AA (10 μM) for 18 h. Levels of invasion are proportional to fluorescence detected by a bottom reading BMG FLUOstar OPTIMA plate reader at 488/520 nm (excitation/emission filter). Data represent means±s.e.m. of three separate experiments (in triplicate). *P<0.05, **P⩽0.01 vs no Y27632 towards TCP or AA, respectively. (B) P-MLC2 levels in PC3-GFP cells incubated with AA and/or the ROCK inhibitor Y27632. Lysates of serum-starved PC3-GFP cells incubated with AA (10 μM)±Y27632 (40 μM, 1 h pre-incubation) at different times were subjected to western blotting. Phosphorylation levels of MLCt18/s19 were compared with GAPDH. Figure is representative of two separate experiments done in duplicate. (C) Invasion assays with PC3-GFP cells were performed using cell-culture inserts (8 μm pore size) coated by a synthetic basement membrane (Matrigel) (left panel) or culture inserts (8 μm pores) coated by a layer of BMEC above a synthetic basement membrane (Matrigel) (right panel). 2 × 105 PC3-GFP cells were added to the top of the inserts and allowed to invade towards AA (10 μM) for 18 h. PC3-GFP cells were transfected with specific RhoA (siRhoA), RhoC (siRhoC), ROCK1 (siROCK1), ROCK2 (siROCK2) siRNA pools, respectively, or with non-targeted siRNA (siNT) 2 days before the invasion assays. Data represent means±s.e.m. of three separate experiments (in triplicate). **P⩽0.01 vs siNT towards AA. (D) Invasion assays with DU-145 cells were performed using cell-culture inserts (8 μm pore size) coated by a synthetic basement membrane (Matrigel) (left panel) or culture inserts (8 μm pores) coated by a layer of BMEC above a synthetic basement membrane (Matrigel) (right panel). 2 × 105 DU-145 cells were added to the top of the inserts and allowed to invade towards AA (10 μM) for 18 h. DU-145 cells were transfected with specific RhoA (siRhoA) and RhoC (siRhoC) siRNA pools, respectively, or with non-targeted siRNA (siNT) 2 days before the invasion assays. Data represent means±s.e.m. of three separate experiments (in triplicate). *P<0.05, ** P⩽0.01 vs siNT towards AA. Cells were counted after staining with either crystal violet or CK immunostaining.