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  • br indicating augmentation of adhesion of cells to matrix The


    indicating augmentation of adhesion of cells to matrix. The same effect was observed in TGF-β/siCofilin-1 cells, with increased packing and size of focal adhesions compared with that of control cells (Fig. 7A–C).
    4. Discussion
    CRC is one of the most common malignant tumors worldwide; however, despite the benefits of early diagnosis and standardized sur-gical treatment, the overall survival of this cancer type remains un-satisfactory [2]. Malignant tumor cells acquire the ability to migrate and invade as a result of the EMT, in which cells can initially lose and/ or disorganize the cell–cell adhesion system and reorganize their PD98059 cytoskeleton [4]. In this context, cofilin-1, an actin-binding protein and dynamic actin cytoskeleton regulator, plays a key role in the main-tenance of cellular activities, and various evidence has demonstrated its crucial role in cancer progression [7,9,15]. However, there is little knowledge regarding the molecular mechanisms underlying cofilin-1 during EMT in CRC.
    To explore the involvement of cofilin-1 in the EMT development of CRC, we employed moderately differentiated human colon cancer cells, HT-29, which were treated with TGF-β, a well-known EMT inducer. Initially, we verified the regulation of the cofilin-1 pathway during EMT in our model. Consistent with the finding that RhoA-LIMK2-cofilin-1 signaling activated by TGF-β is involved in actin cytoskeletal re-organization with stress fiber formation [13,23], our data showed that TGF-β induces RhoA-p-LIMK2-p-cofilin-1 activation in our study model after 48 h of treatment. Moreover, we verified that the inhibitor SB431542 significantly reverted RhoA signaling, decreasing LIMK2 and cofilin-1 phosphorylation, which was in agreement with previous
    Fig. 4. Effects of cofilin-1 silencing on switch between epithelial and mesenchymal states in TGF-β-treated cells. (A) HT-29 cells were transfected with si-CTR or siCofilin-1 and untreated or treated with TGF-β for 48 h. Western blotting and densitometric analysis of E-cadherin, claudin-3, and vimentin are shown. GAPDH was used as a loading control. Bar graphs represent relative quantification of protein expression. Data are presented as the mean ± SEM of three independent experiments. Significance was determined using ANOVA followed by the Bonferroni post-test (*P < 0.05, **P < 0.01,
    (B) Representative immunofluorescence images of E-cadherin and claudin-3 obtained by confocal microscopy of HT-29 cells transfected with si-CTR or siCofilin-1 and treated with TGF-β for 48 h. Fluorescence images of Cy3–vimentin localization are also shown. Scale bar: 10 μm.
    observations [25,26]. Furthermore, TGF-β signaling induced phos-phorylation of cofilin-1, consequently increasing actin polymerization as demonstrated by the augmented F/G-actin ratio, generating an ac-cumulation of filaments and therefore decreasing the G-actin pool. It is important to note that F-actin is responsible for creating and driving the motor force for cellular movement as well as contraction and retraction of the rear of the cell [24].
    Subcellular localization analysis by immunofluorescence of total and p-cofilin-1 and the organizational pattern of actin in TGF-β-treated cells showed distinct subcellular distribution in our EMT model as 
    compared with untreated cells. We further confirmed this result using super-resolution images. Total cofilin-1 was located in the leading edge of the membrane where rapid actin turnover is required for lamelli-podium growth, while p-cofilin-1 was increased throughout the cell in close proximity to stress fibers, indicating the need of spatial control of cofilin-1 activity in EMT cells. Cofilin-1 is a terminal effector of Rho GTPase signaling, and it is widely known that RhoA and Rac have an-tagonistic activities spatiotemporally separated. While RhoA is involved in the initial formation of leading edge protrusion/retraction dynamics, Rac1 and Cdc42 are responsible for activation of pathways involved in