Fasudil

Hek279T, MDA-MB-231, LM2, BT549, Cal51, Cal120 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 9% fetal bovine serum (Sigma), 2mM glutamine, 0.1mg/ml penicillin and 0.1ml/ml streptomycin (Gibco). HCC1806, HCC38, Hs578T cells were maintained in RPMI supplemented with glutamine.
Hek293T cells were used for lentivirus production as described previously [19 (link)]. shRNAs targeting EGFR and ROCK were obtained from the TRCs1.0 library and were as follows: shEGFR-1: TRCN0000121068, shEGFR-2: TRCN0000010329, shEGFR-3: TRCN0000121206, shEGFR-4: TRCN0000121203, shROCK1-1: TRCN0000002163, shROCK1-2: TRCN0000121316, shROCK1-3: TRCN0000121095, shROCK1-4: TRCN0000002161 (TRC Library, Sigma).
For long-term cell growth assays, cells were seeded on 6-well or 12-well plates (Corning). Drugs were added on the following day and media was refreshed every third day with new compound dilutions. At the end time point, the cells were stained with crystal violet. ROCK inhibitors used were GSK269962A (Axon) and Fasudil (Selleck). EGFR inhibitors used were Gefitinib (MedChem) and Afatinib (Selleck).
For DNA content and cell cycle analysis, sub-confluent cells were incubated with 10uM Bromdeoxyuridine (BrdU) for 1.5 hours, trypsinized, fixed in 70% ice-cold ethanol, and stained with anti-BrdU and Propidium Iodide (PI).

Fasudil (FAS, HA-1077, Selleck Chemicals Inc. Houston, USA) was dissolved in saline (1 μg/µL). To investigate the therapeutic effect of Fasudil on CSR, Fasudil and Fasudil + CSR mice were intraperitoneally injected with Fasudil (10 mg/kg) once daily for 14 successive days, 7 days before the CSR protocol. Control mice and CSR mice were intraperitoneally injected with an equivalent amount of vehicle solvent. All injections were carried out at 3:00 P.M. to avoid any effects due to changes in pharmacokinetics and were continued until the last day of CSR. After the injection, the control and Fasudil mice were returned to their cages in another room, and the CSR and Fasudil + CSR mice were maintained in the CSR box. Immediately after CSR, behavioural tests were evaluated, and the brain tissue was harvested for further tests.

All pharmacological treatments were performed in artificial cerebral spinal fluid (aCSF): 125 mM NaCl, 2.5 mM KCl, 26.2 mM NaHCO₃, 1 mM NaH₂PO₄, 11 mM glucose, 5 mM Hepes, 2.5 mM CaCl₂, 1.25 mM MgCl₂, and 0.2 mM APV. Neuronal cultures were pre-treated with inhibitor compounds for 30 min prior to application of Dkk1 recombinant protein (R&D systems). Fasudil (SelleckChem) were dissolved in water and used at concentrations indicated in the text.

Nitroglycerin (NTG) (Beijing Reagent, China) was prepared from a stock solution of 5.0 mg/ml dissolved in water, 30% propylene glycol, and 30% alcohol. For the injections, NTG was freshly diluted to a dose of 1 mg/ml with saline, containing 6% propylene glycol and 6% alcohol. The diluted NTG was administered intraperitoneally (i.p.) at a dose of 10 mg/kg every other day for 9 days (i.e., days 1, 3, 5, 7, and 9) [28 (link)]. An equivalent volume of saline, 6% propylene glycol, and 6% alcohol was used as the vehicle.
To detect the precise role of P2Y12R in CM, animals were injected i.p. with the selective P2Y12 antagonists MRS2395 (1.5 mg/kg; Sigma-Aldrich, MO, USA) and clopidogrel (15, 45, and 100 mg/kg; Selleck, TX, USA) five times, every other day prior to NTG injections. MRS2395 and clopidogrel were dissolved in DMSO solution, which was used as the vehicle. To examine the role of RhoA/ROCK2 in CM, animals were injected i.p. with the ROCK2 inhibitor fasudil (30 mg/kg; Selleck, TX, USA), which was diluted with saline. fasudil was administered in the same manner as MRS2395 and clopidogrel. The concentrations and drug delivery methods of MRS2395, clopidogrel, and fasudil were selected based on previous research [19 (link), 29 (link)–32 (link)].

Antibodies to RhoA, S6K, TSC2, p190RhoGAP, TIAM1, IQGAP, HA-Tag, and Myc-Tag were purchased from Santa Cruz and antibodies to actin, Flag, and Phospho-70S6K were from Life Sciences. Anti-ANDV Gn monoclonal antibody was purchased from United States Biologicals. Anti-N-protein polyclonal rabbit sera made to NY-1V N protein was previously described (25 (link), 26 (link)), and RhoA-glutathione S-transferase (GST) activation assays were performed with GST-Rhotekin-RBD from Cytoskeleton Inc. Bradykinin was purchased from Sigma, and fasudil and Y27632 were purchased from Selleck Chemicals.