Rho Activator (Cat. # CN01) is useful for efficient activation of RhoA, RhoB, and RhoC in a variety of cultured cells.  The reagent activates Rho proteins in fibroblasts, neurons, epithelial, endothelial, and hematopoietic cells as well as other primary and immortalized lines.  Cells treated with the activator can be subjected to any one of a number of assays that indicate an increase in Rho activity, including focal adhesion or stress fiber staining (Cat. # BK005) and Rho activity assays by G-LISA™ (Cat. # BK124).  See Figure 1 for example of Rho activation measured by the G-LISA assay. 

There are many activators of RhoA,B and C proteins in mammalian cells. Commonly used ones are calf serum (1), lysophospatidic acid (LPA)(1) and calpeptin (2). Through years of experience in Rho activation assay, Cytoskeleton Inc. has identified calpeptin as a compound that activates many cell types and has a long timespan of activation for ease of use so this is the active component in Rho Activator Cat.# CN01. Note: Calpeptin is used as an inhibitor of calpain, but it also inhibits myosin light chain phosphorylation which is connected to stress fiber formation and hence possibly to RhoA activation. 

Biological Activity

The effects of CN01 can be mitigated by the use of a Rho Inhibitor such as Cat. # CT04, a reagent that efficiently inactivates cellular Rho proteins in as little as 2 h.  Unit Definition: Using CN01 at 1 unit / ml will activate Rho by 1.5 to 3 fold in epithelial, endothelial, hematopoietic and primary human cell types as measured by the G-LISA^TM Rho Activation Assay (see Figure 1) and observed by stress fiber formation (see Figure 2).

Figure 1. Activation of RhoA in Swiss 3T3 cells by CN01 and LPA.   Cells were grown in DMEM plus 10% fetal calf serum for 2 days, followed by 1% serum for 20h and 0% serum for 16h. Then activators were added to the media and cultures harvested in G-LISA lysis buffer at each time point. RhoA activity was measured with the RhoA G-LISA Activation Assay (Cat. # BK124) and OD490nm plotted on the graph. LPA time course in magenta squares, and CN01 in blue diamonds. Note: The LPA signal is very short lived whereas the CN01 time course is broad which allows full activation to be measured as the experimental control.

The concentration of Rho Activator required for efficient activation of Rho proteins can vary between cell types and whether the medium contains serum or not.  In addition, the length of treatment can be manipulated to yield a moderate or robust phenotype (see Table 1).  For these reasons, the concentration of this reagent and the duration of treatment should be determined by the user.  Typically the effective range is between 0.25 units / ml and 1.0 unit/ml for incubation in serum free medium. In media containing serum it might be difficult to observe the difference between CN01 treated versus untreated samples because there are activators in the serum added to cultured cells.  Inconjunction, incubation times of 5 to 10 min create a moderate phenotype and 10 to 20 min create a robust phenotype.  Recommended conditions for several cell types are detailed in Table 1.

Table 1.  Suggested Conditions for Rho activation in serum free medium.  The indicated cells were subjected to Rho activation assays with CN01 in serum free medium. Serum containing medium will cause activation in all samples so overriding the effects seen by CN01 alone.  Optimal conditions were determined by manipulating reagent concentration and the duration of treatment.

Note: A moderate phenotype is characterized by a 30-40% increase in Rho activity accompanied by stress fiber formation (see Figures 1 and 2). A robust phenotype is characterized by >200% increase in Rho activity accompanied by a stress fiber formation.

Figure 2. Rho Activator (CN01) induces actin stress fibers in serum starved 3T3 cells.   Swiss 3T3 fibroblasts plated on coverslips at 1000 cells / cm2 and grown for two days in DMEM plus 10% fetal calf serum at 37°C and 5% CO2,  were serum starved for 1 day at 1% serum and 1 day at 0% serum. Cultures were treated with 5 µl of CN01 per ml of medium for 10min at 37°C.  Cells were then fixed, stained with rhodamine-labeled phalloidin (Cat. # PHDR1 or BK005), and visualized by fluorescence microscopy.  Images were taken at a magnification of 40×.  The untreated control cells were treated with 5ul DMSO per ml of medium.  The cells treated with CN01 prodcued abundant stress fibers whereas control had less than 10% of CN01 levels of stress fibers (A and B respectively). Under similar conditions the activity of Rho increased by 50% as measured by the G-LISATM RhoA Activation Assay (Cat.# BK124).

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