FIGURE 4:
Moesin shRNA knockdown attenuates actin stress fiber assembly and full morphological transition during TGF-β–induced EMT. (A) Immunoblots of lysates from NMuMG cells treated with TGF-β, showing changes in protein expression during EMT. Wild-type (WT), control shRNA, and moesin shRNA cells were maintained in the absence (–) or presence (+) of TGF-β for 48 h. Blots were probed with antibodies for E-cadherin, N-cadherin, moesin, and ezrin. Blots were also probed with antibodies for β-actin and α-tubulin as loading controls. (B) E-cadherin immunolabeling and F-actin staining of cells, showing changes in organization of cell–cell adhesions and actin cytoskeleton during EMT. Wild-type, control shRNA, and moesin shRNA cells were maintained in the absence (–) or presence (+) of TGF-β (5 ng/ml) for 48 h. Also shown are wild-type cells cotreated with the ROCK inhibitor Y-27632. Fixed cells were labeled with antibodies for E-cadherin and with rhodamine-phalloidin. Arrows indicate cells having fewer, thinner, or absent actin stress fibers. Bar, 20 μm. (C) Quantitative analysis of cell morphology of TGF-β–treated cells in B, showing that the degree of elongated cell morphology, or morphological index, is reduced in moesin shRNA knockdown cells. Horizontal lines indicate the mean value for each cell type. Representative data shown are from a single experiment, for which n was between 30 and 40 for each cell type. Similar data were obtained from two independent cell preparations. **p < 0.01 by one-way ANOVA followed by Dunnett's post test. (D) F-actin staining, showing impaired assembly of actin stress fibers in moesin shRNA knockdown cells. Control shRNA and moesin shRNA cells were maintained in the absence (−) or presence (+) of a low concentration of TGF-β (1.25 ng/ml) for 24 h. Fixed cells were stained with rhodamine-phalloidin. Bar, 20 μm.