Figure 5.
Androgens activate Moesin and actin remodeling through androgen and estrogen receptors in T47D cells. T47D cells were treated with 10−7M T, DHT, or DHEA for 20 min, in the presence or absence of the AR inhibitor flutamide (FLUT, 10−6M), the ER antagonist ICI 182,780 (ICI, 10−6M), the aromatase inhibitor aminoglutethimide (AG, 10−6M), the G protein inhibitor pertussis toxin (PTX, 100 ng/mL), and the Rho-kinase inhibitor Y-27632 (RKI, 10−5M). (A–C) Whole cell extracts were resolved by SDS-PAGE and wild-type Moesin, and Thr558-phosphorylated Moesin (p-Moesin) levels were analyzed by western blot. Images are representative of triplicate experiments. Representative images are shown, and the bar graph shows the mean ± SEM of the ratio Moesin/p-Moesin optical density relative to control of three independent experiments (#p ≤ 0.05 versus each treatment). (D) Actin filaments were stained with phalloidin linked to Texas Red (red staining), and nuclei were counterstained with DAPI (blue staining). Immunofluorescence analysis reveals the dynamic modifications of actin fibbers localization and the formation of specialized cell membrane structures. The box on top of the cells display the intensity of the signal throughout the sample areas measure (two per cell, indicated as the white line). Images are representative, and the bar graph represents the mean ± SEM of the membrane/cytosol actin fluorescent intensity ratio relative to control of triplicate experiments (#p ≤ 0.05 versus each treatment).