Rho Activator II

Rho Activator II

Product Uses Include

  • Activator for Rho pathway
  • Study the effects of Rho activation on cell motility
  • Study the effects of Rho activation on the rearrangement of the actin cytoskeleton
  • Investigate the effects of Rho activation with respect to cross talk with other signal transduction pathways

      The G-switch™ Direct Modulators have been developed with an emphasis on creating highly potent cell permeable reagents that directly target the Rho family of small G-proteins.  Our Direct Activator reagents are based on the catalytic domain of the bacterial CNF toxins, which are covalently attached to a proprietary cell penetrating moiety.  Rho Activator II (Cat# CN03) enters the cell and activates Rho GTPase isoforms by deamidating glutamine-63, which is located in the Switch II region of these GTPases (1).  This modification converts glutamine-63 to glutamate, which blocks intrinsic and GAP stimulated GTPase activity resulting in constitutively active Rho (2).  Rho Activator II robustly increases the level of GTP bound RhoA within 2-4 h after addition to the culture medium, providing a more rapid alternative to transfection based methods for introducing activators like GEFs into cells.  Moreover, the targeted action of Rho Activator II makes it far more attractive tool for the study of Rho GTPase signaling than classic indirect activators (e.g. LPA) that concomitantly activate other signaling pathways (e.g. Ras, PI3K and PLC).



      • Schmidt, G., et al., Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1. Nature, 1997. 387(6634): p. 725-9.
      • Flatau, G., et al., Toxin-induced activation of the G protein p21 Rho by deamidation of glutamine. Nature, 1997. 387(6634): p. 729-33.

      For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com

      AuthorTitleJournalYearArticle Link
      Xu, Weiyi et al.Paxillin promotes breast tumor collective cell invasion through maintenance of adherens junction integrityMolecular biology of the cell2022ISSN 1939-4586
      Chi, Yinxiu et al.Deficiency of Integrin β4 Results in Increased Lung Tissue Stiffness and Responds to Substrate Stiffness via Modulating RhoA ActivityFrontiers in Cell and Developmental Biology2022ISSN 2296-634X
      Hilfenhaus, Georg et al.A High-Content Screen Identifies Drugs That Restrict Tumor Cell Extravasation across the Endothelial BarrierCancer Research2021ISSN 1538-7445
      Wang, Yuan et al.LIMD1 phase separation contributes to cellular mechanics and durotaxis by regulating focal adhesion dynamics in response to forceDevelopmental Cell2021ISSN 1878-1551
      Eddy, Christopher Z. et al.Morphodynamics facilitate cancer cells to navigate 3D extracellular matrixScientific Reports2021ISSN 2045-2322
      Gaston, Cécile et al.EpCAM promotes endosomal modulation of the cortical RhoA zone for epithelial organizationNature Communications2021ISSN 2041-1723
      Kim, Nayoung et al.Mitf promotes cell growth, migration and invasion in clear cell renal cell carcinoma by activating the rhoa/yap signal pathwayCancers2021ISSN 2072-6694
      Li, Wenhong et al.Differential cellular responses to adhesive interactions with galectin-8- And fibronectin-coated substratesJournal of Cell Science2021ISSN 1477-9137
      Hoffmann, Lena et al.Cofilin1 oxidation links oxidative distress to mitochondrial demise and neuronal cell deathCell Death and Disease2021ISSN 2041-4889
      Saraswathibhatla, Aashrith et al.Coordinated tractions increase the size of a collectively moving pack in a cell monolayerExtreme Mechanics Letters2021ISSN 2352-4316
      Laly, Ana C. et al.The keratin network of intermediate filaments regulates keratinocyte rigidity sensing and nuclear mechanotransductionScience Advances2021ISSN 2375-2548
      Talwar, Shefali et al.Mechanosensitive smooth muscle cell phenotypic plasticity emerging from a null state and the balance between Rac and RhoCell Reports2021ISSN 2211-1247
      Wegscheid, Michelle L. et al.Patient-derived iPSC-cerebral organoid modeling of the 17q11.2 microdeletion syndrome establishes CRLF3 as a critical regulator of neurogenesisCell Reports2021ISSN 2211-1247
      Fox, Megan E. et al.Dendritic remodeling of D1 neurons by RhoA/Rho-kinase mediates depression-like behaviorMolecular Psychiatry2020ISSN 1476-5578
      Pan, Meng et al.BNIP-2 retards breast cancer cell migration by coupling microtubule-mediated GEF-H1 and RhoA activationScience Advances2020ISSN 2375-2548
      Hernandez, Paula A. et al.Actomyosin contractility confers mechanoprotection against tnfα-induced disruption of the intervertebral discScience Advances2020ISSN 2375-2548
      Bhosle, Vikrant K. et al.SLIT2/ROBO1-signaling inhibits macropinocytosis by opposing cortical cytoskeletal remodelingNature Communications2020ISSN 2041-1723
      Thomas, Minnah et al.Desmosomal Junctions Govern Tissue Integrity and Actomyosin Contractility in Apoptotic Cell ExtrusionCurrent Biology2020ISSN 1879-0445
      Xia, Mingyu et al.Activation of the RhoA-YAP-β-catenin signaling axis promotes the expansion of inner ear progenitor cells in 3D cultureStem Cells2020ISSN 1549-4918
      Flasse, Lydie et al.Apical Restriction of the Planar Cell Polarity Component VANGL in Pancreatic Ducts Is Required to Maintain Epithelial IntegrityCell Reports2020ISSN 2211-1247
      Rong, Zhouyi et al.Activation of FAK/Rac1/Cdc42-GTPase signaling ameliorates impaired microglial migration response to Aβ42 in triggering receptor expressed on myeloid cells 2 loss-of-function murine modelsFASEB Journal2020ISSN 1530-6860
      Cheong, Sek Shir et al.The Planar Polarity Component VANGL2 Is a Key Regulator of MechanosignalingFrontiers in Cell and Developmental Biology2020ISSN 2296-634X
      Yong, Yu et al.Regulation of degenerative spheroids after injuryScientific Reports2020ISSN 2045-2322
      Hosseini, Kamran et al.EMT-Induced Cell-Mechanical Changes Enhance Mitotic Rounding StrengthAdvanced Science2020ISSN 2198-3844
      Adeniba, Olaoluwa O. et al.Simultaneous time-varying viscosity, elasticity, and mass measurements of single adherent cancer cells across cell cycleScientific Reports2020ISSN 2045-2322
      Hellinger, Johanna W. et al.Identification of drivers of breast cancer invasion by secretome analysis: insight into CTGF signalingScientific Reports2020ISSN 2045-2322
      Yu, Sun Min et al.Mechanical Adaptations of Epithelial Cells on Various Protruded Convex GeometriesCells2020ISSN 2073-4409
      Fujiwara, Sachiko et al.Disease-associated keratin mutations reduce traction forces and compromise adhesion and collective migrationJournal of cell science2020ISSN 1477-9137
      Stankiewicz, Trisha R. et al.Corrigendum to “Dysregulation of Rac or Rho elicits death of motor neurons and activation of these GTPases is altered in the G93A mutant hSOD1 mouse model of amyotrophic lateral sclerosis” [Neurobiology of Disease 136 (2020) 104743] (Neurobiology of DiseaNeurobiology of Disease2020ISSN 1095-953X
      Lundin, Vanessa et al.YAP Regulates Hematopoietic Stem Cell Formation in Response to the Biomechanical Forces of Blood FlowDevelopmental Cell2020ISSN 1878-1551
      Rangel, Laura et al.Caveolin-1α regulates primary cilium length by controlling RhoA GTPase activityScientific Reports2019ISSN 2045-2322
      Eliazer, Susan et al.Wnt4 from the Niche Controls the Mechano-Properties and Quiescent State of Muscle Stem CellsCell Stem Cell2019ISSN 1875-9777
      López-Posadas, Rocío et al.Inhibiting PGGT1B Disrupts Function of RHOA, Resulting in T-cell Expression of Integrin α4β7 and Development of Colitis in MiceGastroenterology2019ISSN 1528-0012
      Mosier, Jenna A. et al.Extent of Cell Confinement in Microtracks Affects Speed and Results in Differential Matrix StrainsBiophysical Journal2019ISSN 1542-0086
      Zhao, Meng et al.Enhancing the engraftment of human induced pluripotent stem cell-derived cardiomyocytes via a transient inhibition of rho kinase activityJournal of Visualized Experiments2019ISSN 1940-087X
      Yong, Yu et al.p75NTR and DR6 regulate distinct phases of axon degeneration demarcated by spheroid ruptureJournal of Neuroscience2019ISSN 1529-2401
      Mistriotis, Panagiotis et al.Confinement hinders motility by inducing RhoA-mediated nuclear influx, volume expansion, and blebbingJournal of Cell Biology2019ISSN 1540-8140
      Dias Gomes, Martim et al.Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrityNature Communications2019ISSN 2041-1723
      Kalinski, Ashley L. et al.Deacetylation of Miro1 by HDAC6 blocks mitochondrial transport and mediates axon growth inhibitionJournal of Cell Biology2019ISSN 1540-8140
      Shu, Liang et al.Brain ischemic insult induces cofilin rod formation leading to synaptic dysfunction in neuronsJournal of Cerebral Blood Flow and Metabolism2019ISSN 1559-7016
      Zhang, Qiao et al.Mechanical Stabilization of the Glandular Acinus by Linker of Nucleoskeleton and Cytoskeleton ComplexCurrent Biology2019ISSN 0960-9822
      Kalappurakkal, Joseph Mathew et al.Integrin Mechano-chemical Signaling Generates Plasma Membrane Nanodomains that Promote Cell SpreadingCell2019ISSN 1097-4172
      Miao, Hui et al.Cell ratcheting through the Sbf RabGEF directs force balancing and stepped apical constrictionJournal of Cell Biology2019ISSN 1540-8140
      Wenceslau, Camilla F. et al.Formyl peptide receptor-1 activation exerts a critical role for the dynamic plasticity of arteries via actin polymerizationPharmacological Research2019ISSN 1096-1186
      Rafiq, Nisha Bte Mohd et al.A mechano-signalling network linking microtubules, myosin IIA filaments and integrin-based adhesionsNature Materials2019ISSN 1476-4660
      Huang, Yuxing et al.Arp2/3-branched actin maintains an active pool of GTP-RhoA and controls RhoA abundanceCells2019ISSN 2073-4409
      Zanotelli, Matthew R. et al.Energetic costs regulated by cell mechanics and confinement are predictive of migration path during decision-makingNature Communications2019ISSN 2041-1723
      Rafiq, Nisha Bte Mohd et al.Forces and constraints controlling podosome assembly and disassemblyPhilosophical Transactions of the Royal Society B: Biological Sciences2019ISSN 1471-2970
      Roxas, Jennifer Lising et al.Enteropathogenic Escherichia coli EspH-Mediated Rho GTPase Inhibition Results in Desmosomal PerturbationsCellular and Molecular Gastroenterology and Hepatology2018ISSN 2352--345X
      Vite, Alexia et al.Α-Catenin-Dependent Cytoskeletal Tension Controls Yap Activity in the HeartDevelopment (Cambridge)2018ISSN 1477-9129
      Werner, Craig T. et al.E3 Ubiquitin-Protein Ligase SMURF1 in the Nucleus Accumbens Mediates Cocaine SeekingBiological Psychiatry2018ISSN 1873-2402
      Herbert, Lindsay M. et al.RhoA increases ASIC1a plasma membrane localization and calcium influx in pulmonary arterial smooth muscle cells following chronic hypoxiaAmerican Journal of Physiology - Cell Physiology2018ISSN 1522-1563
      Rahman-Zaman, Aniqua et al.Cell Migration in Microfabricated 3D Collagen Microtracks is Mediated Through the Prometastatic Protein GirdinCellular and Molecular Bioengineering2018ISSN 1865-5033
      Shiu, Jau Ye et al.Nanopillar force measurements reveal actin-cap-mediated YAP mechanotransductionNature Cell Biology2018ISSN 1476-4679
      Han, Han et al.Regulation of the Hippo Pathway by Phosphatidic Acid-Mediated Lipid-Protein InteractionMolecular Cell2018ISSN 1097-4164
      Kapfhamer, Joshua et al.The Gα q/11 -provoked induction of Akr1c18 in murine luteal cells is mediated by phospholipase CMolecular and Cellular Endocrinology2018ISSN 1872-8057
      Freeman, Spencer A. et al.Transmembrane Pickets Connect Cyto- and Pericellular Skeletons Forming Barriers to Receptor EngagementCell2018ISSN 1097-4172
      Tsuchiya, Masaki et al.Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formationNature Communications2018ISSN 2041-1723
      Jones, Matthew C. et al.Cell adhesion is regulated by CDK1 during the cell cycleJournal of Cell Biology2018ISSN 1540-8140
      Huang, Chao et al.Tetraspanin-enriched microdomains regulate digitation junctionsCellular and Molecular Life Sciences2018ISSN 1420-9071
      Hirata, Hiroaki et al.Actomyosin contractility provokes contact inhibition in E-cadherin-ligated keratinocytesScientific Reports2017ISSN 2045-2322
      Daoud, Abdelkader et al.Molecular and functional crosstalk between extracellular Hsp90 and ephrin A1 signalingOncotarget2017ISSN 1949-2553
      Wu, Mengrui et al.Gα13 negatively controls osteoclastogenesis through inhibition of the Akt-GSK3β-NFATc1 signalling pathwayNature Communications2017ISSN 2041-1723
      Benedicto, Ignacio et al.Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factorsNature Communications2017ISSN 2041-1723
      Li, Yao et al.Rho kinase activity governs arteriolar myogenic depolarizationJournal of Cerebral Blood Flow and Metabolism2017ISSN 1559-7016
      Widmeier, Eugen et al.A small molecule screening to detect potential therapeutic targets in human podocytesAmerican Journal of Physiology - Renal Physiology2017ISSN 1522-1466
      Bade, Nathan D. et al.Curvature and Rho activation differentially control the alignment of cells and stress fibersScience Advances2017ISSN 2375-2548
      Lázaro-Diéguez, Francisco et al.Cell-cell adhesion accounts for the different orientation of columnar and hepatocytic cell divisionsJournal of Cell Biology2017ISSN 1540-8140
      Hamze-Komaiha, Ola et al.SHIP2 Regulates Lumen Generation, Cell Division, and Ciliogenesis through the Control of Basolateral to Apical Lumen Localization of Aurora A and HEF 1Cell Reports2016ISSN 2211-1247
      Park, Yong Hwan et al.Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDSNature Immunology2016ISSN 1529-2916
      Yu, Yonghao et al.Hydrogen-rich medium ameliorates lipopolysaccharide-induced barrier dysfunction via rhoa-mdia1 signaling in caco-2 cellsShock2016ISSN 1540-0514
      López-Posadas, Rocío et al.Rho-A prenylation and signaling link epithelial homeostasis to intestinal inflammationJournal of Clinical Investigation2016ISSN 1558-8238
      Agbor, Larry N. et al.Cullin-3 mutation causes arterial stiffness and hypertension through a vascular smooth muscle mechanismJCI Insight2016Article Link
      Otsu, Keishi et al.The Semaphorin 4D-RhoA-Akt Signal Cascade Regulates Enamel Matrix Secretion in Coordination With Cell Polarization During Ameloblast DifferentiationJournal of Bone and Mineral Research2016ISSN 1523-4681
      Sayedyahossein, Samar et al.Integrin-Linked Kinase Is Indispensable for Keratinocyte Differentiation and Epidermal Barrier FunctionJournal of Investigative Dermatology2016ISSN 1523-1747
      Jackson, William D. et al.Very-low and low-density lipoproteins induce neutral lipid accumulation and impair migration in monocyte subsetsScientific Reports2016ISSN 2045-2322
      Li, Shufeng et al.Microtopographical features generated by photopolymerization recruit RhoA/ROCK through TRPV1 to direct cell and neurite growthBiomaterials2015ISSN 1878-5905
      Freeman, Spencer A. et al.Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptorNature Communications2015ISSN 2041-1723
      Lahne, Manuela et al.Actin-cytoskeleton-and rock-mediated inm are required for photoreceptor regeneration in the adult zebrafish retinaJournal of Neuroscience2015ISSN 1529-2401
      Lee, Meng Horng et al.Normal mammary epithelial cells promote carcinoma basement membrane invasion by inducing microtubule-rich protrusionsOncotarget2015ISSN 1949-2553
      Justus, Calvin R. et al.GPR4 decreases B16F10 melanoma cell spreading and regulates focal adhesion dynamics through the G13/Rho signaling pathwayExperimental Cell Research2015ISSN 1090-2422
      Aifuwa, Ivie et al.Senescent stromal cells induce cancer cell migration via inhibition of RhoA/ROCK/myosin-based cell contractilityOncotarget2015ISSN 1949-2553
      Bhandary, Lekhana et al.ROCK inhibition promotes microtentacles that enhance reattachment of breast cancer cellsOncotarget2015ISSN 1949-2553
      Takito, Jiro et al.Regulation of Osteoclast Multinucleation by the Actin Cytoskeleton Signaling NetworkJournal of cellular physiology2015ISSN 1097--4652
      Herr, Michael J. et al.Tetraspanin CD9 regulates cell contraction and actin arrangement via RhoA in human vascular smooth muscle cellsPLoS ONE2014ISSN 1932-6203
      Lavall, Daniel et al.The mineralocorticoid receptor promotes fibrotic remodeling in atrial fibrillationJournal of Biological Chemistry2014ISSN 1083-351X
      De Vega, Susana et al.A C-terminal fragment of fibulin-7 interacts with endothelial cells and inhibits their tube formation in cultureArchives of Biochemistry and Biophysics2014ISSN 0003-9861
      Sun, Shan et al.Impact of oxidative stress on cellular biomechanics and rho signaling in C2C12 myoblastsJournal of biomechanics2014ISSN 1873--2380
      Valtcheva, Nadejda et al.The orphan adhesion G protein-coupled receptor GPR97 regulates migration of lymphatic endothelial cells via the small GTPases RhoA and Cdc42Journal of Biological Chemistry2013ISSN 0021-9258
      Pignatelli, Jeanine et al.Actopaxin (α-parvin) phosphorylation is required for matrix degradation and cancer cell invasionJournal of Biological Chemistry2012ISSN 0021-9258
      Kuang, Shao Qing et al.Rare, nonsynonymous variant in the smooth muscle-specific isoform of myosin heavy chain, MYH11, R247C, alters force generation in the aorta and phenotype of smooth muscle cellsCirculation Research2012ISSN 0009-7330
      Iyer, Padma et al.Connective tissue growth factor-mediated upregulation of neuromedin U expression in trabecular meshwork cells and its role in homeostasis of aqueous humor outflowInvestigative Ophthalmology and Visual Science2012ISSN 0146-0404

      Question 1:  Can the direct Rho activator CN03 be used with cells growing in culture?

      Answer 1:  Yes, CN03 is specifically designed to be used as a Rho activator with cultured cells.  The active site of CN03 is based on the catalytic domain of the bacterial cytotoxic necrotizing factor (CNF) toxins.  The catalytic domain of CN03 is covalently attached to a proprietary cell penetrating moiety.  Upon entry into the cell, CN03 activates Rho GTPase isoforms by deamidating glutamine-63, which is located in the Switch II region of these GTPases.  This modification converts glutamine-63 to glutamate, which blocks intrinsic and GAP-stimulated GTPase activity, resulting in constitutively active Rho.  CN03 robustly increases the level of GTP-bound RhoA within 2-4 h after addition to the culture medium.  


      Question 2:  How can I assess whether Rho activity is changing in my cells following CN03 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). 


       If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com.