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Supplementary figure legends, BJC MD/2013/3583R

FIGURE LEGEND OF SUPPLEMENTARY FIGURES:

Figure 1: Amoeboid characteristics of PC3-GFP cells in presence of AA

Time lapse picture of PC3-GFP cells incubated with AA. PC-3 cell morphology visualized over time by brightfield phase microscopy, after loading with 10µM AA. Scale bar = 50µm. Cells started to retract 2.9±0.6 to 14.6±3.0 minutes after the addition of AA, became visibly smaller and began to round up. This was followed by membrane blebbing 26.7±4.5 minutes post treatment and 69.7±3.2 minutes after the addition of AA cells began to round up fully, a process completed within 92.2±2.3 minutes.

Figure 2: PC-3 phenotype during TEM

A. Time lapse microscopy of PC-3 during TEM. The cell (arrowhead) migrates through the bone marrow endothelial (BMEC) monolayer. In the images from 01:44 to 04:31 the cell can be seen migrating across the substrate underneath BMEC. Times are shown as hours and minutes after the first image at 00:00. Scale bar represents 100µm. See also video clip.

B. Electronic images of PC-3 cells during TEM, incubated with AA. Scanning electron micrograph of a BMEC monolayer 5 hours after addition of PC-3 cells. BMEC are labelled B and PC-3 cells are labelled 1 and 2. Upper panel: flat view of the specimen from above showing both PC-3 cells. Lower left: the same specimen tilted to around 60° showing an improved view of the pseudopodium of PC-3 cell number 1 Lower right: the area indicated by the box of lower left panel; the PC-3 cell pseudopodium (black arrowhead) can be seen extending into the BMEC cell-cell junction (white arrowheads). Upper panel) x2000, lower left) x2000, Lower right) x3000 magnification.

C. BMEC permeability of fluorescent dextran in presence of specific Rho/ROCK and Rac inhibitors. Confluent monolayers of BMEC were cultured on fibronectin coated Transwell chambers with 1µm diameter pores. PC-3 cells at 1x105 cells per well or inhibitors (to a final concentration in the chamber of Y-27632 at 40µM, blebbistatin at 75µM, Rac1 inhibitor at 75µM or GM6001 at 20µM) were added to the culture medium in the Transwell chamber and incubated for 6 hours. After incubation with inhibitors or PC-3 cells, 12.5µl FITC-dextran (molecular weight 40,000) at 1mg/ml in PBS was added to the Transwell chamber and allowed 30 minutes to diffuse through the BMEC monolayer into the lower chamber before the level of fluorescence in the lower chamber was read (BMG Labtech). A series of standard dilutions of FITC-dextran in medium were made to allow the amount of diffusion through the BMEC monolayer to be calculated (data not shown).

A plain Transwell insert without fibronectin or BMEC allowed 4.6% of the dextran to diffuse into the lower well, while a fibronectin coated Transwell chamber allowed 7.3% to diffuse; both results were significantly higher than diffusion through the BMEC monolayer (p<0.05 for both results). Once a BMEC monolayer was present, diffusion of dextran was reduced to 1.5% and the presence of PC-3 cells or inhibitors did not significantly affect monolayer permeability (p>0.05 for PC-3 cells and all inhibitors).

BMEC control - no inhibitors; FN control - fibronectin coated transwell insert without BMEC or inhibitors; transwell control - transwell insert without fibronectin, BMEC or inhibitors. Results are shown as mean of 3 experiments, bars indicate means ± SEM.

Figure 3: Effects of Y27632 and H1152 on PC3-GFP

A. PC-3 cell viability, evaluated by Trypan blue coloration, in the presence of escalating doses of Y27632 (n=3). All analyses carried out 18 hours post dosing. Data represent means of 3 separate experiments (in triplicate).

B. PC3-GFP morphology in presence of Y27632. Microscopic analysis of PC3-GFP cells incubated with AA (10mM). Serum starved PC3-GFP cells were incubated with AA (10mM) for 3 hours, the vehicle only during 3 hours (Ctrl), or preincubated with Y27632 (40mM) 1 hour prior to the addition of AA (10mM). Cells were then fixed, coloured with crystal violet and photographed.

C. Adhesion assays were performed using PC3-GFP cells which were either preincubated with Y27632 (40mM) 1 hour before the assay or in vehicle control. 4x104 PC3-GFP cells were then incubated with or without AA (10mM) for 8min with a confluent layer of BMEC plated in 96 wells. Wells were washed twice in PBS. Levels of adhesion are proportional to fluorescence detected by a bottom reading BMG FLUOstar OPTIMA plate reader at 488/520nm (excitation/emission filter). Data represent means ± SEM of 3 separate experiments. ** P≤ 0.01 vs vehicle only with AA.

D. H1152 inhibition of ROCK reduces transendothelial migration of PC3-GFP cells towards arachidonic acid. PC3-GFP cells were serum starved over night at 37°C, 5% CO2. Cells were treated with either 2.5mM or 5mM H1152 for 30 minutes prior to addition to a transwell chamber containing a Matrigelä or a BMEC/Matrigelä barrier at 105 cells/well. Cells were allowed to invade towards 10mM AA overnight in the presence of H1152.

The addition of the ROCK inhibitor H1152 had no effect on AA induced invasion through a Matrigelä barrier. However increasing doses of H1152 inhibited PC3-GFP cell invasion towards AA through a BMEC/Matrigelä without inducing loss of cell viability (data not shown).

Figure 4: Effects on TEM of combination knock-down of Rho signalling components in PC3-GFP cells towards AA.

Invasion assays with PC3-GFP cells were performed using cell culture inserts (8µm pore size) coated by a layer of BMEC above a synthetic basement membrane (Matrigel™). 2x105 PC3-GFP cells were added to the top of the inserts and allowed to invade towards AA (10mM) for 18h. PC3-GFP cells were transfected with specific RhoA (siRhoA), RhoC (siRhoC), ROCK1 (siROCK1), ROCK2 (siROCK2) siRNA pools respectively, with combination of siRho A and siRhoC, siROCK1 and siROCK2, or with non-targeted siRNA (siNT) 2 days before the invasion assays. Data represent means ± SEM of 2 separate experiments (in triplicate). ** P≤ 0.01 vs siNT towards AA.

Figure 5: PC3-GFP morphology incubated with or without AA and with selective knock-down of the Rho/ROCK pathway

Microscopic analysis of serum starved PC3-GFP cells incubated with or without AA (10mM) for 3 hours. PC3-GFP cells were transfected previously with specific RhoA (siRhoA), RhoC (siRhoC), ROCK1 (siROCK1), ROCK2 (siROCK2) siRNA pools respectively or with non-targeted siRNA (siNT) 2 days before the beginning of the experiment. Cells were visualised by brightfield phase microscopy. Scale bar is 50mm.

Figure 6: Selective RhoA and RhoC knock-down in DU-145 cells.

Western blot analysis of DU-145 cells transfected for 48h with specific RhoA (siRhoA) and RhoC (siRhoC) siRNA pools respectively, or transfected with non-targeted siRNA (siNT). Loading was evaluated by GAPDH. Figure is representative of two independent experiments.