Figure 3.

Transfection studies with RhoA ⁺ and RhoA ⁻ plasmid constructs, showing that RhoA modulates SM myogenesis by modulating mesenchymal cell shape. Undifferentiated mesenchymal cells were isolated from E11 mouse embryonic lungs and transfected with the two different RhoA plasmid constructs before spreading (1 h after plating). (A) Cells transfected with RhoA⁺ and immunostained with anti-HA1 tag remain round in shape (top left). Spread nontransfected cells in the same picture cannot be visualized because they are not stained and are in a different focus. Round cells do not immunostain for desmin (bottom left; arrow points to one of these cells). In the same picture, note desmin-positive spread cells (arrowhead points to one of them). Cells transfected with RhoA⁻ have spread (top right) and demonstrate strong immunopositivity for desmin (bottom right). (B) Immunoblots of cells transfected with RhoA⁺ and RhoA⁻. Mesenchymal cells transfected with RhoA⁻ spread faster than the nontransfected cells in the same culture, leading to an increase in SM gene expression. For comparison, mitogen-activated protein kinases show no differences in levels. (C and D) RhoA⁺-transfected mesenchymal cells, which detach faster than spread nontransfected counterparts, were collected from the cultures by short trypsinization and immediately lysed. (C) After 24 h, cells expressing RhoA⁺ have the same RhoA activity as control undifferentiated mesenchymal cells 1 h after isolation. Here RhoA activity assay was run on 3.5 μg of protein per lane. (D) Cells expressing RhoA⁺ do not undergo SM myogenesis as indicated by the absence of SM myosin, desmin, and SM α-actin. Note that 3 μg protein/lane was loaded for this experiment rather than the 15 μg protein loaded in other experiments. Thus, the bands seen in the vector control sample are less intense than in other panels. Bar, 20 μm.