Figure 8. Actin remodeling by estrogen receptor requires Gα₁₃, RhoA and ROCK-2.

A) T47-D cells transiently transfected with either empty vector or with plasmids encoding for constitutively active or dominant-negative Gα₁₃ (Gα₁₃ CA or Gα₁₃ DN) or RhoA (RhoA CA or RhoA DN) were treated for 15 min with 10 nM E2. Other cells also received a co-treatment with E2 (10 nM; 15 min) and the ROCK inhibitor Y-27632 (Y; 10 µM). Breast cancer cell actin fibers were stained with phalloidin linked to Texas Red, and nuclei were counterstained with DAPI. B) shows the analytic results obtained by using Leica QWin image analysis and processing software showing the mean thickness of the cell membrane as well as the intensity of actin staining in the cytoplasm, membrane or extracellular compartment. The results are derived from the sampling of five areas of the cell membrane of thirty different random cells. The areas of minimum and maximum cell membrane thickness were always included. The results are the mean±SD of the measurements. * = p<0.05 vs. vehicle-transfected control. C) shows the changes of the amount of filamentous actin (F-actin, F) content versus free globular-actin (G-actin, G) content in T47-D cells after treatment with E2 (10 nM) for 15 min, in the presence or absence of ER antagonist ICI 182,720 (ICI; 1 µM), G protein inhibitor PTX (100 ng/ml) and ROCK-2 inhibitor Y-27632 (Y; 10 µM). Positive (Pos) and negative (Neg) controls were set by adding F-actin enhancing solution (phalloidin, 1 µM) or F-actin depolymerization solution (10 µM cytochalasin-D) to the lysates, respectively.