Product Uses Include
This product consists of highly purified C3 Transferase (Cat. # CT03) covalently linked to a proprietary cell penetrating moiety via a disulfide bond. The cell penetrating moiety allows rapid and efficient transport through the plasma membrane. Once in the cytosol, the cell penetrating moiety is released, thereby allowing C3 Transferase to freely diffuse intracellularly and inactive RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1.
The Exoenzyme C3 Transferase from Clostridium botulinum is commonly used to selectively inactivate the GTPases RhoA, RhoB, and RhoC, both in vivo and in vitro. C3 Transferase inhibits Rho proteins by ADP-ribosylation on asparagine 41 in the effector binding domain of the GTPase. A major limitation of C3 Transferase for use in in vivo applications is that this protein is only slightly cell permeable. Consequently, overnight incubations with C3 Transferase at concentrations as high as 100 µg/ml are often necessary to inactivate Rho proteins in cultured cells. Under these conditions, the long incubation period and large amount of C3 Transferase that are required can be disruptive to basic cellular functions and economically burdensome. Cytoskeleton, Inc. has overcome these problems by providing a cell permeable form or C3 Transferase that can efficiently inactivate cellular Rho proteins in as little as 2 h.
CT04 has been used to inactive Rho proteins to an efficiency of 75-95% in fibroblasts, neurons, epithelial, endothelial, and hematopoietic cells as well as other primary and immortalized cell lines (see Table 1 for recommended conditions of use for different cells types).
|Table 1. Suggested Conditions for Rho inactivation by Cell Permeable C3 Transferase. The indicated cells were subjected to Rho inactivation assays with CT04. Note: These concentrations were determined in serum free medium. For cells grown in serum containing medium the recommended concentration is four times higher.|
2.0 µg/ml, 2 h (1, 2)
2.0 µg/ml, 4-6 h (1, 2)
0.5 µg/ml, 2 h (1, 2)
0.5 µg/ml, 4-6 h (1, 2)
1.0 µg/ml, 2 h (1, 2)
1.0 µg/ml, 4-6 h (1, 2)
1.0 µg/ml, 2 h (1, 2)
1.0 µg/ml, 4-6 h (1, 2)
1.0 µg/ml, 2 h (2)
1.0 µg/ml, 4 h (2)
1.0 µg/ml, 4 h (2)
1.0 µg/ml, 4-6 h (1, 2)
* A moderate phenotype is characterized by a 10-40% decrease in Rho activity accompanied by stress fiber disruption and a well spread morphology (see Figures 1 and 2).
** A robust phenotype is characterized by a >50% decrease in Rho activity accompanied by a loss of stress fibers, cell body collapse, and protrusion of dendrite-like extensions (see Figures 1 and 2).
1 Based on morphological assays examining cell shape and the architecture of the actin cytoskeleton.
2 Based on biochemical data from Rho activity assays.
The Exoenzyme C3 Transferase from Clostridium botulinum has been produced in a bacterial expression system. The recombinant protein contains six histidine residues at its amino terminus (His tag), has a molecular weight of approximately 24 kDa, and is greater than 90% pure (see Cat. # CT03 for gel purity). To make the purified C3 Transferase protein cell permeable, a proprietary cell penetrating moiety has been linked via a reversible disulfide bond. Cell Permeable Rho Inhibitor is supplied as a white lyophilized powder.
Cell Permeable Rho Inhibitor (Cat. # CT04) is useful for efficient inactivation of RhoA, RhoB, and RhoC in a variety of cultured cells. The reagent inhibits Rho proteins in fibroblasts, neurons, epithelial, endothelial, and hematopoietic cells as well as other primary and immortalized lines. Cells treated with Cell Permeable C3 Transferase can be subjected to any one of a number of assays that indicate a decrease in Rho activity, including focal adhesion or stress fiber (Cat. # BK005) disruption assays and Rho activity assays by G-LISA™ (Cat. # BK124) or pulldown (Cat. # BK036). See Figures 1 and 2 for examples of stress fiber disruption and Rho inactivation demonstrating CT04 biological activity.
Figure 1. Cell permeable Rho inhibitor disrupts stress fibers and can be manipulated to induce either moderate or robust phenotypes. Swiss 3T3 fibroblasts plated on coverslips were untreated (A) or treated with 2.0 µg/ml of CT04 for 2 h (B) or 4 h (C) at 37°C. Cell were then fixed, stained with Rhodamine-labeled Phalloidin (Cat. # BK005), and visualized by flu orescence microscopy. Images were taken at a magnification of 20×. The untreated control cells in A were well spread and stress fibers were present. The cells treated for 2 h in B displayed a Moderate Phenotype, characterized by a loss of stress fibers, cells remaining well spread, and a 10-40% decrease in Rho activity (also see Figure 2). Treatment for 4 h (C) yielded a Robust Phenotype, characterized by a loss of stress fibers, decreased cell spreading, collapse of the cell body, protrusion of dentritic extensions, and a >50% decrease in Rho activity (also see Figure 2).
Figure 2a. CT04 inhibition of Rho activity as measured with the RhoA G-LISA Activation Assay (Cat.# BK124). Serum starved Swiss 3T3 fibroblasts were untreated (no CT04) or trea ted with 0.20, 0.50 and 2.0 µg/ml of CT04 for 4h in serum free medium at 37°C, then activated with 100µg/ml calpeptin for 10min. Cells were then lysed and RhoA activity was measured by the RhoA G-LISA Activation Assay (Cat.# BK124). Note: At 2.0 µg/ml CT04 for 4h results in almost complete (90%) inhibition of RhoA activity.
Figure 2b. Cell permeable Rho Inhibitor decreases RhoA activity. Swiss 3T3 fibroblasts were untreated (no CT04) or treated with 2.0 µg/ml of CT04 for 2 h (CT04, 2 h) or 4 h (CT04, 4 h) at 37°C. Cells were then lysed and RhoA activity was measured by pulldown assay using the Rho-binding domain of the Rho effector Rhotekin (RhoA Activation Assay Biochem Kit, Cat. # BK036). The pulldowns (active RhoA) and cell extracts (total RhoA) were analyzed by SDS-PAGE followed by Western blotting with a RhoA specific antibody. The level of RhoA activity in each sample is proportional to the amount of RhoA precipitated in the pulldowns (active RhoA, upper panel).
M.J. Herr et al., 2014. Tetraspanin CD9 regulates cell contraction and actin arrangement via RhoA in human vascular smooth muscle cells. PLoS ONE. 9:3106999.
Sakai et al., 2013. LPA1-induced cytoskeleton reorganization drives fibrosis through CTGF-dependent fibroblast proliferation. FASEB J. doi: 10.1096/fj.12-219378.
Ginestier et al., 2012. Mevalonate metabolism regulates basal breast cancer stem cells and is a potential therapeutic target. Stem Cells. 30, 1327-1337.
Zhang et al., 2012. Self-Assembling Peptide Nanofiber Scaffold Enhanced with RhoA Inhibitor CT04 Improves Axonal Regrowth in the Transected Spinal Cord. J. Nanomaterials. doi:10.1155/2012/724857.
Kim et al., 2012. Inhibition of RhoA but not ROCK induces chondrogenesis of chick limb mesenchymal cells. Biochem. Biophys. Res. Comm. 418, 500-505.
Wan et al., 2012. Regulation of myosin activation during cell–cell contact formation by Par3-Lgl antagonism: entosis without matrix detachment. Mol. Biol. Cell. 23, 2076-2091.
Balzer et al., 2012. Physical confinement alters tumor cell adhesion and migration phenotypes. FASEB J. doi: 10.1096/fj.12-211441.
Kakudo et al., 2011. The effect of C3 transferase on human adipose-derived stem cells. Hum. Cell. 24, 4165-169.
Fan et al., 2011. Macrophage Migration Inhibitory Factor and CD74 Regulate Macrophage Chemotactic Responses via MAPK and Rho GTPase. J. Immunol. 186 4915-4924.
Kim et al., 2009. Statins decrease dendritic arborization in rat sympathetic neurons by blocking RhoA activation. J. Neurochem. 108, 1057-1071.
Martinelli et al., 2009. ICAM-1–mediated endothelial nitric oxide synthase activation via calcium and AMP-activated protein kinase is required for transendothelial lymphocyte migration. Mol. Biol. Cell.20, 995–1005.
Question 1: Can the Rho inhibitor CT04 be used with cells growing in culture?
Answer 1: Yes, Cytoskeleton modified the exoenzyme C3 Transferase from Clostridium botulinum to be cell permeable (Cat. # CT04) for the specific purpose of inhibiting Rho in living cells in as little as 2 hours.
The exoenzyme C3 Transferase from Clostridium botulinumis commonly used to selectively inactivate the GTPases RhoA, RhoB, and RhoC, both in vivoand in vitro. C3 Transferase inhibits Rho proteins by ADP-ribosylation on asparagine 41 in the effector binding domain of the GTPase. A major limitation of the standard C3 Transferase for use in in vivoapplications is that this protein is not cell permeable.
Cytoskeleton’s cell permeable Rho inhibitor consists of highly purified C3 Transferase covalently linked to a proprietary cell penetrating moiety via a disulfide bond. The cell penetrating moiety allows rapid and efficient transport through the plasma membrane. Once in the cytosol, the cell penetrating moiety is released, thereby allowing C3 Transferase to freely diffuse intracellularly and inactive RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1.
Question 2: How can I assess whether Rho activity is changing in my cells following CT04 treatment?
Answer 2: There are multiple ways to measure changes in Rho activity. To visualize a change in Rho activity, we recommend examining Rho-mediated stress fiber formation with fluorescently-labeled phalloidin (Cat. # PHDG1, PHDH1, PHDN1, PHDR1). These Acti-stain phalloidins label F-actin stress fibers. Activation of Rho can be directly quantified with one of our activation assays, either the traditional pull-down (Cat. # BK036) or the RhoA G-LISA activation assay (Cat. # BK124).
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