br Cofilin depletion rescues partial
3.2. Cofilin-1 depletion rescues partial epithelial morphology of cells in EMT through cytoskeletal rearrangements
Actin organization is a key factor in the generation and maintenance of cell morphology and polarity . To investigate whether cofilin-1 is required to switch from epithelial to fibroblast-like morphology, we inhibited cofilin-1 expression in HT-29 cells with siRNA. Cofilin-1 protein expression was inhibited by approximately 70% after 48 h of treatment with siRNA (Fig. 3A). Next, we measured the changes in cell morphology through circularity and cell body area analysis. Fluores-cence images were utilized for measuring the Lovastatin area, which de-lineates the area of the cell. In this experiment, control cells showed a cuboid shape similar to a perfect circle (equal to 1), but cells with va-lues distant from 1 were more fibroblast-like. The circularity of the TGF-β-treated cells (0.49 ± 0.16) was lower than that of control cells (0.76 ± 0.10), and the cell body area analysis demonstrated increased cell size, reflecting the membrane projection as lamellipodia as well increased nuclear and cytoplasmic volume in these cells. Silenced co-filin-1 cells treated with TGF-β (siCofilin-1/TGF-β) showed partial re-storation of cuboid cell morphology (0.66 ± 0.15) as well as partial restoration of cell size (Fig. 3B). These findings suggested that cofilin-1 is required to establish the fibroblast-like morphology during EMT through cytoskeletal rearrangements. Therefore, we next hypothesized that cells exposed to TGF-β could have increased actin polymerization through cofilin-1, consequently leading to the formation of stress fibers. However, siCofilin-1/TGF-β cells did not show inhibition of actin polymerization, since the actin filament length analysis revealed in-creased F-actin packaging (Fig. 3C), suggesting actin filament in-stability. Indeed, biochemical fractionation of TGF-β-stimulated cells demonstrated equal portions of F-actin in cofilin-1-depleted and control cells, although decreased G-actin pool was observed in cells depleted of cofilin-1, indicating impaired depolymerization (Fig. 3D). Taken to-gether, these data suggested that cofilin-1 has a significant role in driving actin dynamics, reflected by the actin stability and regulation of cell morphology during EMT. r> 3.3. Cofilin-1 is required for switching between epithelial and mesenchymal states and junctional disassembly during EMT
The cortical F-actin belt of intestinal cells anchors at TJs and AJs, driving their assembly and function . To evaluate if cofilin-1 could regulate the disassembly of the cell–cell adhesion system during EMT, we analyzed the expression and subcellular localization of epithelial and mesenchymal proteins in siCofilin-1/TGF-β cells. As expected, in cells treated with TGF-β, the E-cadherin and claudin-3 protein levels were decreased relative to those in control cells; however, cells treated with siCofilin-1/TGF-β exhibited restoration of E-cadherin and claudin-3 at cell-cell contact. In addition, the claudin-3 protein levels but not of E-cadherin were restored in these cells. Consistently, the TGF-β group showed increased vimentin. Nevertheless, with concomitant depletion
Fig. 1. TGF-β activates RhoA/p-LIMK2/p-cofilin-1 signaling and increases F/G-actin ratio in HT-29 cell line. (A) Representative RhoA pull-down assay for detection of RhoA-GTP with Rhotekin RBD. HT-29 cells were grown and treated with TGF-β (10 ng/mL) for 48 h. The precipitate and lysate were used to determine RhoA-GTP and total RhoA levels by western blot using anti-RhoA antibody following a pull-down assay.
(D) Cells were treated with TGF-β (10 ng/mL) for 48 h, and then total fractionation of F-actin and G-actin was performed and quantified as described in Materials and methods. As a control, treatment with 20 nM of cytochalasin D was used. Representative images are shown. GAPDH was used as a loading control in (B) and (C). Bar graphs represent relative quantification of p/LIMK2/LIMK2 and p/cofilin1/cofilin1 ratio (where non-treated cells = 1). 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, ***P < 0.001).
of cofilin-1, the vimentin protein levels were decreased, and the vi-mentin filament network was reorganized (Fig. 4A and B). Additionally, actin aggregates in close proximity to cytoplasmic E-cadherin deposits in the TGF-β group were observed, suggesting that the arrangement and/or dynamics of cortical actin filaments contributed to the
disassembly of junctions, whereas SB43154 inhibitor restored the actin and E-cadherin organization at AJCs (Supplementary Fig. 3). These data suggested that cofilin-1 signaling is required for switching between epithelial and mesenchymal states and for junctional disassembly by regulating the dynamics of the actin cytoskeleton.