Simvastatin sim

All chemicals were of the highest quality available (e.g., analytical grade). Bovine calf serum and 3T3-L1 mouse embryonic fibroblasts and were purchased from the American Type Culture Collection (ATCC, USA). Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide (MTT), and N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid were obtained from Gibco Invitrogen (Grand Island, NY, USA). Insulin solution (bovine), 3-isobutyl-1-methylxanthine (IBMX), 4-nitrophenyl dodecanoate (pNP laurate), porcine pancreas lipase, and simvastatin (SIM) were obtained from Sigma-Aldrich (St. Louis, USA). Dimethyl sulfoxide (DMSO) was purchased from Carlo Erba Reagents S.r.l. (Chaussée du Vexin, Val de Reuil, USA). Dexamethasone was obtained from G Bioscience (St. Louis, USA). Oil Red O was purchased from Amresco Inc. (Solon, OH, USA).

K₄[Fe(CN)₆], bovine serum albumin(BSA), MnCl₂, 2’,7’-dichlorofluorescin-diacetate (DCF-DA), Oil Red O (ORO), lipopolysaccharide (LPS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydrogen peroxide assay kit (MAK 165) were purchased from Sigma-Aldrich (U.S.A.). 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 2,2,6,6-Tetramethylpiperidine 1-oxyl (TEMP) and simvastatin (Sim) were purchased from Sigma-Aldrich (Shanghai). (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG) was obtained from Corden Pharma (Switzerland). Cyanine5 NHS ester (Cy5) and cyanine7.5 NHS ester (Cy7.5) were from Lumiprobe (U.S.A.). Penicillin, streptomycin, fetal bovine serum (FBS), and Dulbecco’s Modified Eagle's Medium (DMEM) were purchased from Gibco (U.S.A.). 4’,6-Diamidino-2-phenylindole (DAPI), hematoxylin and 3,3’-dioctadecyloxacarbocyanine perchlorate (DiO) were purchased from Beyotime (China). LysoTracker Green was purchased from Invitrogen (U.S.A.). Recombinant murine interferon-γ (INF-γ) was purchased from PeproTech (U.S.A). Laser confocal scanning microscopy (CLSM)-specific dishes (35 mm × 10 mm) were purchased from Corning Inc (New York).

African green monkey kidney (Vero; ATCC CCL-81) cells were grown in growth medium consisting of Minimal Eagle’s Medium (MEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, antimycotic antibiotics (Gibco), and ʟ-glutamine (Gibco). ZIKV (ATCC VR-1838) was propagated in Vero cells using MEM supplemented with 0.3% bovine serum albumin (BSA, Gibco), antimycotic antibiotics, MEM vitamins (Gibco), and ʟ-glutamine (Infection Medium). The ZIKV particles were harvested at 5 days postinfection (dpi), and culture supernatants were stored at −80 °C until use. ZIKV titers were determined through the plaque assay in Vero cells.
Atorvastatin calcium salt trihydrate (ATO), fluvastatin sodium hydrate (FLU), and simvastatin (SIM) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Mevastatin sodium salt (MEV) was purchased from Calbiochem (San Diego, CA, USA). Statin stocks (20 mM) were prepared in DMSO and stored in aliquots at −20 °C. EZ-Cytox was purchased from DoGenBio, Co., Ltd. (Seoul, South Korea).

Simvastatin (SIM) and mevastatin (MEV) were purchased from Sigma-Aldrich (St. Louis, MI, USA). All flavonoids (acacetin, ACAC; chrysin, CHR;8-prenylnaringenin, 8-PR; 6-prenylnaringenin, 6-PR) and xanthohumol (Xant) were purchased from Alexis Biochemicals. Their chemical structures are presented in Figure 8. Stock solutions of all compounds were prepared in dimethyl sulfoxide (DMSO).

The statins (mevalonate pathway inhibitors) used were simvastatin (SIM, Sigma-Aldrich, Hamburg, Germany), rosuvastatin calcium (ROSU, SelleckChem, Munich, Germany), and atorvastatin calcium salt trihydrate (ATO, Sigma-Aldrich, Hamburg, Germany). Stocks were prepared in DMSO. Primary antibodies for Western Blot analyses were: anti-Rap1A (sc-1482; recognizes the ungeranylated Rap1A^{47 (link)}), anti-HMGCR (sc-271595) from Santa Cruz (Heidelberg, Germany), anti-HMGCR (ab214018) from Abcam (Cambridge, UK), anti-cleaved PARP (#9541) from Cell Signaling Technology, Inc. (Beverly, MA, USA), and anti-SREBP2 (#557037) from BD Biosciences (Heidelberg, Germany). For GAPDH, we used the anti-GAPDH (sc-25778) from Santa Cruz (Heidelberg, Germany) and anti-GAPDH (#5G4) from HyTest Ltd. (Turku, Finland). Secondary horseradish peroxidase-conjugated antibodies were anti-mouse IgG (HAF007), anti-rabbit IgG (HAF008), and anti-goat IgG (HAF109) from R&D Systems, Inc. (Minneapolis, MN, USA).

PLGA (50:50; Resomer^® RG505) was supplied by Boehringer Ingelheim (Ingelheim, Germany). Poly vinyl alcohol (PVA, molecular weight: 13,000–23,000, 98% hydrolyzed), dichloromethane (DCM), gelatin from porcine skin, simvastatin (SIM), and LPS were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cellulose-ester dialysis membrane (MWCO; 6–8 kDa) was obtained from Spectrum Laboratories (Milpitas, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), and penicillin-streptomycin were obtained from Gibco (Rockville, MD, USA).