of Brock et al. [38]. TRITC-labeled 10,000-MW lysine-fixable dextran (Molecular Probes) was used as a marker of loaded cells. Coverslips with cell monolayers were transferred into new dishes. Aliquots (10 ml) of protein solution (0.5 mg/ml) mixed with 1 ml of dextran were added on the monolayer surface. Monolayers were wounded with an injection needle, rinsed with warm DMEM, and incubated in DMEM before fixation with 3.7% paraformaldehyde (PFA). RNA interference To deplete mDia1 in cells, we used the RNAi technique. The small interfering RNA oligonucleotides, corresponding to two mDia1 sequences from positions 795 to 813 and 184 to 209, were described previously [29] and chemically synthesized by Syntol (Moscow, Russia). Synthetic siRNA duplexes based on sense and antisense sequences were prepared as described [39]. GFP siRNA was used as negative control. We transfected mDia1 siRNA or control siRNA duplex oligonucleotides into cells using Lipofecta- mine 2000 according to the manufacturer’s protocol (Invitrogen). The cultures were incubated for 36–48 h and analyzed with Western blotting or fixed after wounding of cell monolayers and establishment of new cell-cell contacts. Cell migration assay We determined the migratory capacity of IAR cells in vitro using BD Bio-CoatTM migration chambers containing polycarbonate membrane inserts with 8-mm pores in a 24-well tissue culture plate (Becton Dickinson). Cells (26104 cells/200 ml of DMEM with 8% FCS) were added in the upper wells of chambers. The bottom well of each chamber was filled with DMEM with 8% FCS. After 20 h of incubation at 37uC with 5% CO2, cells from the upper surface of membranes were completely removed with a cotton swab; the migratory cells on the lower surface of membranes were fixed with 100% methanol and stained with DAPI. Membranes were mounted onto glass slides and examined microscopically at 206 magnification. We determined cellular migration by counting the numbers of stained cells on membranes in 15 randomly selected fields. The means of triplicate assays for each cell line in three independent experiments were used. Nude mouse tumorigenicity assay Cells were resuspended in DMEM (1 million cells/300 ml DMEM) and were injected subcutaneously into the right rear flank of athymic nude (nu/nu) mouse (eight mice per group). Animals were monitored weekly for appearance and measurement of tumors. Tumor volumes (V) were determined from the three dimensions (height (a) by width (b) by depth (c) of tumors) (in millimeters) and calculated as V = 1/6pabc according to [40]. Animals were sacrificed 4 weeks postinjection, and the tumors, where present, were analyzed histologically and weighed. Fluorescence staining and microscopy For E- or N-cadherin staining, cells were fixed with methanol- acetone (1:1) at 220uC for 10 min. For double staining of E- or N- cadherin and of actin, cells were fixed with 1% PFA and permeabilized with 0.5% Triton X-100 for 5 min. To visualize mDia1, cells were fixed with 3.7% PFA and permeabilized. In some experiments, fixed cells were heated to 100uC and transferred into fresh PBS. Fixed specimens were incubated for 40 min with primary antibodies and then for 40 min with TRITC- or Alexa488-conjugated secondary antibodies. Alexa488- or TRITC-conjugated phalloidin was added to secondary antibodies. Fixed samples were examined with a Zeiss Axioplan Adhesive Interactions microscope equipped with a 1006(NA 1.4) objective. Images were acquired with a cooled CCD ORCA-ER camera (Hamamatsu Photonics) using Wasabi 1.5 software. We measured the intensity of E-cadherin and N-cadherin fluorescence at cell-cell contacts using ImageJ software (NIH, USA) as described [23]. The average of peak pixel intensity values in square segments (10610 pixels) covering the entire cell-cell contact length was taken as a measure of fluorescence intensity at cell-cell contacts. For the quantification of cadherin accumulation at cell-cell contacts, we calculated a localization index by subtracting the peak value of cytoplasmic intensity from the peak value at the cell-cell contact and dividing this difference by the peak value of cytoplasmic intensity. This index was used to calculate relative fluorescence intensity. Control values were taken as 1. Live-cell imaging and image analysis For live-cell imaging, cells were transferred into phenol red free a-MEM medium (Gibco, Invitrogen) supplemented with 10% FCS and were observed with a Nikon Eclipse-Ti microscope equipped with a Plan-Neofluar 1006 (NA 1.3) DIC objective at 35uC. Images were acquired with an ORCA-ER Hamamatsu camera controlled with NIS-Elements AR2.3 software (Nikon). In some cases, we used Leica TCS SP5 confocal laser scanning microscope. We measured overlapping areas as described [32], using SimplePCI 5.3.1 software (Hamamatsu). To analyze lamellipodial dynamics in the narrow wound, time-lapse sequences were acquired with 3-s intervals for 25–30 min. Using ImageJ software (NIH, USA), we generated kymographs along straight lines drawn in the direction of protrusion. Rates of lamellipodial protrusion and retraction were determined on the basis of slopes produced by advancing leading edges. The mean rates during 5 min periods of observation were used. Using ImageJ software, we measured the distances traveled by individual AJs and calculated the mean rates of AJ movement. The distance map method was used to study the dynamics of AJs. We analyzed selected frames from confocal images of GFP-E-cadherin in living cells in sparse cultures using ImageJ software as described [25]. Binary images produced from grey images were thinned until they formed binary skeletons, which represent central positions of AJs. Distance maps (Di) were created; these are images of the distance to the nearest point from the skeleton. Next, the binary skeletons at times i+1 were overlaid on the distance maps (Di). In the absence of movement, the pixel value on the distance map below the skeleton is zero. If movement occurred between the two images, the pixel value below the skeleton indicates the distance traveled between times i and i+1. The average value of the pixel- to-pixel multiplication of Si+1 and Di quantifies the average displacement of AJs between two successive images. Western blotting SDS-PAGE and Western blotting were performed in minigel PAGE devices (Mini-ProteanH 3 Cell, Bio-Rad). For Western blotting of whole cell lysates, cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 6.8; 2% SDS; 16% glycerol; 1% mercaptoethanol; 0.004% bromphenol blue) including protease inhibitor cocktail (Sigma). Samples (10 or 20 ml) were heated for 5 min at 95uC and used immediately for 10% SDS-PAGE in equal protein concentrations according to the Bio-Rad protocol. Resolved proteins were transferred to PVDF membranes, which were then probed with appropriate primary and peroxidase- conjugated secondary antibodies. We detected blotted protein bands using an ECL Plus kit (Amersham), and we captured PLoS ONE | www.plosone.org 15 November 2009 | Volume 4 | Issue 11 | e8027