Y-27632 dihydrochloride: Selective ROCK1/2 Inhibitor for Cytoskeletal and Stem Cell Studies

Executive Summary: Y-27632 dihydrochloride selectively inhibits ROCK1 (IC₅₀ ≈ 140 nM) and ROCK2 (K_i ≈ 300 nM) with >200-fold selectivity over other kinases, supporting precise modulation of the Rho/ROCK signaling pathway ([https://www.apexbt.com/y-27632-dihydrochloride.html](https://www.apexbt.com/y-27632-dihydrochloride.html)). This compound disrupts Rho-mediated stress fiber formation, alters cell cycle progression (G1→S), and impairs cytokinesis in vitro (Mishra et al., 2024). Y-27632 enhances stem cell viability and organoid formation, and suppresses tumor invasion in preclinical models ([https://mutantidh1-in-1.com/index.php?g=Wap&m=Article&a=detail&id=15967](https://mutantidh1-in-1.com/index.php?g=Wap&m=Article&a=detail&id=15967)). Its high solubility in DMSO, ethanol, and water facilitates flexible experimental design. This article extends prior resources by integrating quantitative benchmarks, cell-type context, and workflow parameters for robust experimental translation.

Biological Rationale

Rho-associated kinases (ROCK1 and ROCK2) are central effectors in the Rho/ROCK signaling pathway. They regulate actin cytoskeleton organization, cell contractility, adhesion, migration, and proliferation (Mishra et al., 2024). Aberrant ROCK activity is implicated in cancer, fibrotic, and neurodegenerative diseases due to its roles in cell motility, invasion, and cytokinesis. In cell biology, precise ROCK inhibition is essential for dissecting cytoskeletal dynamics, stem cell maintenance, and tissue remodeling. The development of selective small-molecule inhibitors enables researchers to interrogate ROCK-driven processes in a controlled, quantitative manner.

Mechanism of Action of Y-27632 dihydrochloride

Y-27632 dihydrochloride is a cell-permeable, ATP-competitive inhibitor targeting the catalytic domains of ROCK1 and ROCK2. It binds with high affinity (IC₅₀ ≈ 140 nM for ROCK1; K_i ≈ 300 nM for ROCK2) and exhibits >200-fold selectivity against unrelated kinases like PKC, MLCK, and PAK (ApexBio product page). By inhibiting ROCK activity, Y-27632 disrupts phosphorylation of downstream targets such as MYPT1 and LIMK, leading to loss of actin stress fibers, impaired focal adhesion, and altered cell shape. This inhibition modulates cell cycle progression, especially transition from G1 to S phase, and can block cytokinetic abscission. The net effect is reduced cell proliferation, migration, and altered cytoskeletal structure. In stem cell cultures, Y-27632 preserves viability by preventing dissociation-induced apoptosis (anoikis).

Evidence & Benchmarks

• Y-27632 dihydrochloride inhibits ROCK1 with an IC₅₀ of ~140 nM and ROCK2 with a K_i of ~300 nM at 25°C, pH 7.4 (ApexBio, product page).
• Displays over 200-fold selectivity for ROCK1/2 versus PKC, cAMP-dependent protein kinase, MLCK, and PAK, as measured by in vitro kinase assays at 10 µM substrate concentration (ApexBio).
• Reduces formation of Rho-mediated actin stress fibers within 30 minutes of treatment at 10 µM in HeLa cells (Mishra et al., 2024).
• Decreases proliferation of prostatic smooth muscle cells in a concentration-dependent manner (0.1–10 µM) in vitro (ApexBio).
• In mouse xenograft models, Y-27632 administration suppresses tumor invasion and reduces metastatic burden (5–30 mg/kg, i.p., daily for 10–21 days) (mutantidh1-in-1.com).
• Enhances viability and expansion of human-induced pluripotent stem cells (hiPSCs) following dissociation (10 µM, 24h) (blebbistatin.com).
• Soluble at ≥111.2 mg/mL in DMSO, ≥17.57 mg/mL in ethanol, and ≥52.9 mg/mL in water at 25°C; warming to 37°C or ultrasonic bath increases dissolution rate (ApexBio).
• Stock solutions stable below –20°C for several months; long-term storage of working solutions is not recommended (ApexBio, reference).

Applications, Limits & Misconceptions

Y-27632 dihydrochloride is widely used for:

• Modulating cytoskeletal organization and stress fiber dynamics in adherent cell cultures.
• Enhancing survival and expansion of stem cells and organoids, especially post-dissociation (mutantidh1-in-1.com).
• Investigating the role of ROCK signaling in tumor invasion, metastasis, and cancer cell migration.
• Studying cytokinesis inhibition and cell cycle progression in various model systems.

For a focused guide on intestinal stem cell applications, see Y-27632 Dihydrochloride: Advanced Applications in Intestinal Stem Cell Biology—the present article extends the cytoskeletal and cancer-relevant context, offering broader mechanistic and workflow parameters.

For practical workflows and troubleshooting, Y-27632 Dihydrochloride: Selective ROCK Inhibitor for Advanced Research provides a hands-on guide; this article adds a comparative analysis of selectivity and solution stability.

Common Pitfalls or Misconceptions

• Y-27632 is highly selective for ROCK1/2 but does not inhibit all Rho GTPase effectors—results cannot be extrapolated to non-ROCK targets (ApexBio).
• Long-term storage of aqueous or ethanol stock solutions (>1 month) leads to loss of potency; fresh preparation is recommended.
• In vivo efficacy depends on bioavailability and dosing regimen; not all tumor models respond equivalently (mutantidh1-in-1.com).
• Does not reverse established cytoskeletal defects in neurodegenerative models where endo-lysosomal dysfunction is upstream of ROCK activation (Mishra et al., 2024).
• Not a substitute for genetic knockout when complete pathway ablation is required.

Workflow Integration & Parameters

Y-27632 dihydrochloride is supplied as a solid and should be stored desiccated at 4°C or below. For working solutions, dissolve at ≥111.2 mg/mL in DMSO, ≥17.57 mg/mL in ethanol, or ≥52.9 mg/mL in water. Warming to 37°C or ultrasonic bath aids dissolution. Stock solutions are stable below –20°C for up to several months; avoid repeated freeze-thaw cycles. In cell culture, typical concentrations range from 1–10 µM, with exposure durations from 30 min (cytoskeletal disruption) to 24 h (stem cell survival). Washout studies confirm that most effects are reversible upon compound removal. For in vivo studies, dosing regimens vary; e.g., 5–30 mg/kg/day intraperitoneally for 10–21 days in mouse xenograft models. Always titrate concentration and duration to cell type, endpoint, and experimental goals (ApexBio).

For an advanced synthesis of translational and stem cell systems, see Y-27632 Dihydrochloride: Precision ROCK Inhibitor for Stem Cell Microenvironments, which this article complements by providing detailed solubility and stability parameters.

Conclusion & Outlook

Y-27632 dihydrochloride is a validated, highly selective ROCK1/2 inhibitor for cytoskeletal, cell cycle, stem cell, and cancer research. Its robust performance, solubility, and selectivity profile make it a gold-standard reagent for dissecting Rho/ROCK signaling in vitro and in vivo. Continued integration with disease-relevant models and advanced culture systems will further clarify its therapeutic and research utility. The A3008 kit offers reliable sourcing for reproducible experiments. For translational perspectives, Strategic ROCK Inhibition: Unleashing the Translational Potential of Y-27632 offers a complementary overview, while this article provides granular, implementation-focused data.