Y 27632 dihydrochloride

The ROCK inhibitors Fasudil hydrochloride, Y-27632 dihydrochloride and GSK-429286 were obtained from Tocris. The following materials were purchased from the indicated commercial sources: PD98059 (MEK inhibitor, New England Biolabs); AG1478 (EGFR inhibitor, Calbiochem); AIIB2 (anti-Integrinβ1 antibody; Aragen Bioscience); mAb225 (anti-EGFR antibody; Oncogene); Blebbistatin (myosin II ATPase inhibitor, Cayman Chemical).

To induce muscle injury and regeneration a sharpened tungsten wire was applied to the myotome of anaesthetized larvae embedded in low melting point agarose as previously described [50 (link)]. Either a single punctate injury to ventral myotome 13, or multiple injuries to myotomes 8–14, were performed depending on whether animals were used for imaging or for extraction of RNA.
Animals were exposed to 50 µM Y-27632 dihydrochloride (Tocris) 6 h prior to commencement of the experiment and throughout the experiment with replacement of drug at 12 h intervals. A 10 mM stock dissolved in DMSO was diluted in E3 medium to 50 µM. Control samples were incubated with 0.005% DMSO.

The control hiPSC line was kindly provided by Dr. Jianyi Zhang (University of Alabama at Birmingham), this hiPSC line was reprogrammed from cardiac fibroblasts of a healthy female donor and modified to overexpress cyclin D2 to enhance the yield of cardiac differentiation (Zhu et al., 2018 (link); Zhao et al., 2021 (link)). To generate mutant hiPSCs, 2 h prior to electroporation, hiPSCs were treated with 10 μM ROCK inhibitor (Y27632 dihydrochloride, Tocris). Cells were then passaged and 250 K cells were then spun down in PBS. Cells were resuspended in P3 Primary Cell Nucleofector™ Solution containing Supplement 1 (Lonza, Switzerland) and 1 μg of chemically modified sgRNA (Synthego, Menlo Park, CA) and 1.5 μg codon optimized BE4 mRNA (TriLink Biotechnologies, San Diego, CA) and placed in the 20 μL cuvette provided and CB-150 was the Amaxa protocol used. Cells were given 10 μM ROCKi for 48 h post electroporation followed by normal culturing conditions. For hiPSC single cell isolation ∼300 single cell suspended iPSC were cultured in a 10 cm dish with 10 μM ROCKi for 48 h. For the next 10 days cells were given daily media changes and then colonies were isolated using a 10 μL pipette to scrape half of the colony for isolated culture and half the colony for genomic DNA isolation.

24 h post-transfection (DIV14), the cultured hippocampal neurons were treated either with vehicle (PBS) or with Aβ_1–42 oligomers at varying concentrations: 10 nM, 50 nM, 100 nM, 500 nM. The NB + medium was replaced with fresh Neurobasal medium without supplements and the peptides were diluted directly into the culture wells. The control wells contained no peptides but an equivalent volume of the vehicle used to dilute the oligomerized peptides (PBS). To interfere with p75^NTR Rho-ROCK signaling, the cell permeable p75^NTR inhibitor TAT-Pep5 Calbiochem (100 nM; Merck Millipore) and the p160 ROCK inhibitor Y-27632 dihydrochloride (10 μM; Tocris Bioscience) were used to pre-treat the neurons for 15 min before the addition of Aβ_1–42 oligomers into the same culture medium. For the analysis of dendritic spine density and morphology, the cultures were fixed after 15 min or 6 h. For the analysis of the F-actin content as well as for the measurement of RhoA activation the neurons were either fixed or lysed 10 min after Aβ_1–42 oligomer addition.