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  • br The activity of the RhoA protein was determined


    The activity of the RhoA protein was determined using a pull-down assay (Thermo Fisher Scientific, #89854) according to the manufac-turer's instructions. HT-29 RNase Inhibitor were seeded in a T-25 flask with DMEM + 10% FBS. Upon reaching about 80% confluence, the assay was performed. Rhotekin RBD bound to glutathione–agarose beads was used to precipitate GTP-bound RhoA from the cell lysate. The active and total Rho were separated by 13% SDS-PAGE and visualized by western blotting using antibody against Rho (1:1000).
    2.8. Cell migration assays
    Wound healing assay was performed as described previously [17]. Briefly, HT-29 cells were seeded in 12-well plates, 4 h before the medium was replaced with DMEM + 1% FBS. Upon reaching about 100% confluence, the cell monolayer was scraped by a sterile pipette tip to create wounds, and HT-29 cells were incubated with or without TGF-β or siRNA for cofilin-1. Cells were photographed immediately after wounding (0 h) and after 24 h of incubation. The lesion area was manually quantified using ImageJ software. The values of cell migra-tion were represented as percentages, with means ± standard error of the mean (SEM) of triplicate assays.
    Phagokinetic track motility assay was also executed as described previously [19]. This assay measures motility of individual cells through the trajectory remaining as a result of phagocytosis of colloidal gold. HT-29 cells (2 × 103) with or without TGF-β and siRNA were seeded in gold sol-coated wells and incubated in DMEM + 2% FBS at 37 °C in 5% CO2 for 20 h. After incubation, the cells were photographed using the Axio Observer.Z1 microscope (Carl Zeiss) equipped with an Axio Cam HRc and Axio Vision Release 8.2 Image Analyzer. For quantification of the motility track area, ImageJ software was used. The tests were performed in duplicate, at least 10 photos of each condition were captured, and three independent experiments were performed.
    HT-29 cells treated with TGF-β and/or cofilin-1 siRNA (6 × 104 cells) or untreated cells were seeded in DMEM + 1% FBS in the upper surface of 8-μm-pore Polycarbonate Membrane Transwell® Inserts (Costar) that were coated with 50 μL of Matrigel® (BD Biosciences). DMEM + 10% FBS was added as a chemoattractant in the lower chamber. After 48 h of incubation, the invaded cells on the lower membrane of the inserts were fixed with ethanol (10 min), stained with 1% violet crystal (10 min), and analyzed and counted under a micro-scope. The tests were performed in duplicate, and three independent experiments were performed.
    Equal amounts of HT-29 cells were seeded in six-well plates with or without TGF-β/siRNA in DMEM + 1% FBS. The conditioned culture medium was collected, and equivalent protein amounts were subjected to SDS-PAGE with 10% (v/v) gels containing 0.2% gelatin (porcine skin, type A, Sigma-Aldrich). After electrophoresis, the gels were wa-shed twice (30 min each time at room temperature) in 10 mM Tris/HCl, pH 8.8 containing 2.5% Triton X-100 and then incubated in activation buffer (5 mM CaCl2, 0.02% NaN3, and 50 mM Tris/HCl, pH 8.0) at 37 °C for 16 h. Then, the gels were stained with Coomassie Brilliant Blue R-250 and destained in 10% (v/v) acetic acid and 40% (v/v) methanol. The gelatinolytic activity of the matrix metalloproteinase was detected as transparent bands on the blue background. The gels were quantified by optical density with ImageJ software. 
    2.11. Immunofluorescence microscopy
    Immunofluorescence assay was performed as previously described [17]. Briefly, cell monolayers were grown on glass coverslips until 70% confluence and treated with or without TGF-β/SB431542/siRNA. The monolayers were washed in RNase Inhibitor PBS containing 100 mM CaCl2 and 100 mM MgCl2, pH 8.0 (PBS-CM) and fixed in methanol for 10 min. Ad-ditionally, the monolayers were washed with PBS and permeabilized and blocked with 0.1% Triton X-100 + 0.2% BSA in PBS for 1 h. The cells were incubated overnight at 4 °C in a humid environment with specific primary antibodies: anti-E-cadherin (1:300) and anti-claudin-3 (1:30). Then, the cells were incubated in blocking solution with ap-propriate Alexa Fluor 488 anti-mouse or Alexa Fluor 546 anti-rabbit secondary antibodies (1:200) for 1 h. Afterward, the coverslips were incubated with DAPI (1:1000) for 1 min, washed, and mounted using propyl gallate (Sigma-Aldrich).