Fluvastatin sodium hydrate

Atorvastatin calcium salt trihydrate (ATO), cerivastatin sodium salt hydrate (CER), fluvastatin sodium hydrate (FLU), pravastatin sodium salt hydrate (PRA), rosuvastatin calcium (ROS), simvastatin (SIM), and chloroquine (CHL) were purchased from Sigma-Aldrich. Lovastatin sodium salt (LOV) and mevastatin sodium salt (MEV) were purchased from Calbiochem. The statins ATO, FLU, PRA, ROS, MEV, LOV, and SIM were reconstituted to 20 mM in dimethyl sulfoxide (DMSO); PRA was reconstituted to 50 mM in DMSO; CER was reconstituted to 20 mM in distilled deionized water. All statins were stored at −20 °C and diluted to working concentrations in infection medium on the same day of use. CHL was dissolved to 100 mM in distilled deionized water, stored at 4 °C, and diluted to the required concentration on the same day of use.
Mouse anti-flavivirus group antigen monoclonal antibody clone D1-4G2-4-15 (4G2 mAb) was purchased from Merck-Millipore (MAB10216) and stored at −20 °C in aliquots prior to use. The secondary antibody, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG, was purchased from Sigma-Aldrich. The cytotoxicity assay kit, EZ-CYTOX, was purchased from DoGenBio, Co., Ltd. in South Korea.

Na₂S, nonahydrate (99.99% purity), glucose, hydrocortisone, insulin, apo-transferrin, sodium selenite, sodium orthovanadate, uridine (cell culture grade), Protease Inhibitor Cocktail for use with mammalian cell and tissue extracts, puromycin, cerulenin, fluvastatin sodium hydrate, doxycycline, RIPA buffer, HPLC grade tert-Butyl methyl ether (MTBE), and chloroform were from Sigma. TOFA and ND-646 were from MedChemExpress. Dulbecco's modified Eagle's medium (DMEM) (with 4.5 g/l glucose, glutamine, and 110 mg/l sodium pyruvate), RPMI 1640 with glutamine, medium 199, fetal bovine serum (FBS), penicillin/streptomycin mixture, trypsin, EDTA, PBS, and Dulbecco's phosphate-buffered saline medium (DPBS) were from Gibco. Geneticin was purchased from Life Technologies. Hepes and LC-MS grade acetonitrile, methanol, water, isopropanol, and ammonium formate were from Fisher, and Eagle's minimal essential medium (EMEM) was from Lonza. [U-¹⁴C]-glucose (263.0 mCi/mmol) and [U-¹⁴C]-glutamine (281.0 mCi/mmol) were from PerkinElmer. EquiSPLASH lipid standard (#330731) was purchased from Avanti Polar Lipids, Inc.

To validate the screening hits, simvastatin (S6196, Sigma), fluvastatin sodium hydrate (SML0038, Sigma) and terbinafine hydrochloride (T8826, Sigma) were added to the cell culture and cell viability was analysed 5 days later using CyQUANT™ Cell Proliferation Assay (C7026, Invitrogen) as per manufacturer’s instructions.
CyQUANT™ Cell Proliferation Assay was also used to determine cell proliferation after PT2385 treatment.

siRNA knockdown of genes CDC42 and PAK5 was performed by using Silencer^® Select pre-designed siRNAs (Thermo Fisher Scientific Inc.). Fluvastatin sodium hydrate (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in water and further diluted with culture media. Vehicle control was generated by using equal amount of water.