Chlorpromazine hydrochloride cpz

Hoechst 33342 dye (#62249) and HCS CellMask blue (#H32720) for immunostaining were purchased from ThermoFisher Scientific (Waltham, MA, USA) and used at 1:10,000 and 1:1000 in PBS, respectively. Mouse monoclonal anti-ZEBOV GP (4F3) and anti-ZEBOV VP40 (3G5) antibodies were obtained from IBT Bioservices (Gaithersburg, MD, USA). Mouse monoclonal anti-ZEBOV NP (10B5 C11 C9 C3), and VP35 (2A11 F12 D7) were obtained by Dr. D. Leung (Wash. U. St. Louis, MI, USA). Rabbit polyclonal anti-Ebola VP30 (GTX134035) was purchased from GeneTex (Irvine, CA, USA). Rabbit polyclonal anti-CAPG antibody (10194-1-AP) was from Proteintech (Rosemont, IL, USA). Mouse monoclonal anti-β-actin (MAB8929) antibody was from R&D systems. The following secondary antibodies were purchased from Thermo Scientific: goat anti-mouse Alexa Flour 488, anti-mouse Alexa Flour 546. 5-(N-ethyl-N- isopropyl) amiloride (EIPA) and chlorpromazine hydrochloride (CPZ) were from Sigma-Aldrich (St. Louis, MO, USA). EIPA was diluted to 10 mM with dimethylsulfoxide. CPZ was diluted to 50 mg/mL with water. All the reagents described here were kept at −40 °C until use.

All reagents were of analytical grade. Trypan blue (0.4%) dye, hydrogen peroxide (H₂O₂) 30% w/w, 2,5-diphenyl-3-(4,5-dimethyl-2-thiazolyl) tetrazolium bromide (MTT), dimethylsulfoxide (DMSO), 2,7-dichlorodihydrofluorescein diacetate (DCF) and chlorpromazine hydrochloride (CPZ, CAS No. 69–09-0) were supplied from Sigma-Aldrich (Madrid, Spain). Dulbecco’s modified Eagle’s medium (DMEM) with and without phenol red, fetal bovine serum (FBS), phosphate buffered saline (PBS), l-glutamine solution (200 mM), trypsin-ethylenediaminetetraacetic acid (EDTA) solution (170,000 U/L trypsin and 0.2 g/L EDTA) and penicillin-streptomycin solution (10,000 U/mL penicillin and 10 mg/mL streptomycin) were acquired from Lonza (Verviers, Belgium). All analytical standards used for liquid chromatography analysis shikimic acid, gallic acid, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, (+)-catechin hydrate, (−)-epicatechin, rutin, hyperoside, naringin, quercitrin, 3,5-di-O-caffeoylquinic acid, rosmarinic acid, cinnamic acid, eugenol and trans-cinnamaldheyde were purchased from Sigma-Aldrich (Milan, Italy). The 75 cm² culture flasks and 96-well plates were obtained from TPP (Trasadingen, Switzerland). HyClone fetal bovine serum (FBS) was purchased from Thermo Scientific (Northumberland, United Kingdom).

Cells were seeded to 24-well PET cell culture inserts, 0.4-μm pore size for 7 days, in a two-chambered system. In this system, barrier cells form a polarized monolayer with tight junctions between cells, allowing access to both apical and basolateral domains. Cells were incubated for 30 min with various transcytosis inhibitors in different concentration, including 10 μM of nystatin (Sigma), 10 μM of chlorpromazine hydrochloride (CPZ, Sigma), 20 μM of dimethyl amiloride (DMA, Sigma), and 10 μM of colchicine (Sigma). Drugs were administered to the cells in both apical and basolateral chambers at identical concentrations. Thereafter, 100 μl of Atto647-ZIKV (2 × 10⁶ copies ml^–1 of virus) was added into the upper chamber. After 4 h incubation with cells, all of the basolateral supernatant was collected. Percent transcytosis was determined by measuring the fluorescence intensity of Atto647N through using a fluorescent plate reader.

VUAA1 (N-(4-ethylphenyl)-2-((4-ethyl-5-(3-pyridinyl)-4H-1,2,4-triazol-3-yl)thio)acetamide) was synthesized by the working group “Mass Spectrometry/Proteomics” of the Max Planck Institute for Chemical Ecology (Jena, Germany). W7 hydrochloride was purchased from Tocris bioscience (Wiesbaden-Nordenstadt, Germany), Chlorpromazine hydrochloride (CPZ) and ethyl hexanoate (99% purity) from Sigma Aldrich (Steinheim, Germany).