Chloroquine phosphate

Succinic anhydride, b-PEI (MW 1,800), b-PEI (MW 250,000), 2-(7-Azabenzotriazol-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU), N-ethyldiisopropylamine (DPIEA) et.al was purchased from Adamas-beta (Shanghai, China). All of solvent were purchased from Macklin (Shanghai, China). Chloroquine Phosphate was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies used for Western Blotting and immunofluorescence including rabbit anti-LC3B, anti-p62, anti-Pgp, goat anti-rabbit IgG (H + L) (HRP, Cora Lite 594) were obtained from Proteintech (Wuhan, China). Alexa fluor 647-labeled goat anti-rabbit IgG (H + L), rabbit anti-CD44, anti-ki67, Ad-GFP-LC3B, Lyso-Tracker Red, TUNEL Apoptosis Assay Kit, acid phosphatase assay kit et.al were purchased from Beyotime (Shanghai, China). All other reagents for western blotting and gel electrophoresis were obtained from Solarbio (Beijing, China).
Targeting human P-gp siRNA sequences:
Sense: 5’-AAGAAGGAAAAGAAACCAACUdTdT-3’;
Anti-sense: 5’-AGUUGGUUUCUUUUCCUUCUUdTdT-3’.
All of siRNA were obtained by GenePharma Co. Ltd. (Shanghai, China).

NP046 (C₂₁H₂₆FNO, MW = 327.4) was synthesized and its HPLC purity was determined to be > 99% (for details of the synthesis see Additional file 1). All chemicals and reagents used in this study were of analytical grade or ACS (American Chemical Society) grade. Ammonium formate (97% pure), sodium acetate (purity > 99%), chloroquine phosphate and PEG400 were purchased from Sigma-Aldrich Gmbh (Steinheim, Germany), formic acid (98 – 100%), tert-butylmethyl ether (purity > 99%) and ethanol (GC > 99.9%) were purchased from Merck KGaA (Darmstadt, Germany), acetonitrile and methanol (all of high-purity grade) were purchased from Honeywell, Burdick & Jackson (Muskegon, MI 49442, USA). Water used to prepare solutions was purified by a Millipore Elix 10 reverse osmosis and Milli-Q^® (Millipore, USA) Gradient A 10 polishing system.

Montelukast sodium (Mon) and chloroquine phosphate were bought from Sigma-Aldrich (St. Louis, MO, USA). Curcumin and digitonin were purchased from MedChemExpress (Monmouth Junction, NJ, USA) and Targetmol (Wellesley Hills, MA, USA), respectively. All compounds were dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C except chloroquine phosphate, which was dissolved in sterilized water. The mouse anti-HCoV-OC43 nucleoprotein monoclonal antibody (mAb) was purchased from Millipore (Temecula, CA, USA).

3,5,3′-triiodothyronine (catalog no. T2877), BAPTA-AM (catalog no. A1076), antimycin A (catalog no. A8674), edelfosine (catalog no. sml0332), and chloroquine phosphate (catalog no. PHR1258) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Neutrophil progenitors were maintained in growth media supplemented with 2% conditioned CHO media containing Stem Cell Factor (SCF) and 0.5 μM β-estradiol (Sigma-Aldrich, E2758, Saint Louis, MO, USA). The CRISPRed neutrophil cell lines were authenticated with WB.
HL-60 (ATCC, ccl240) were propagated and differentiated per manufacturer’s instructions without authentication.
To differentiate into granulocytes, cells were cultured in RPMI-1640 media supplemented with 10% FBS, 100 U/ml penicillin/100 μg/mL streptomycin, 2 mM L-Glutamine, 2% SCF in the presence or absence of 20 ng/mL GCSF (BioLegend, 574602, San Diego, CA, USA) or GCSF and 5 ng/ml GM-CSF (BioLegend, 576302, San Diego, CA, USA), and maintained for either 3 or 5 days before performing experiments. Cells were seeded into 6-well plates at a density of 2 × 10⁶ cells/4 mL.
To examine changes in autophagy, neutrophils were treated with 50 μM Chloroquine phosphate (Sigma-Aldrich, 50-63-5, Saint Louis, MO, USA) or vehicle for 4 h before preparing protein lysates. To examine impact of blocking prenylation, neutrophils were treated with 10 μM Cysmethynil (Cayman chemicals, 14745, Ann Arbor, Michigan, USA) for three days.

Vero cells (CCL-81, ATCC, Manassas, VA, USA), A549-Dual™ cells (a549d-nfis, InvivoGen, San Diego, CA, USA) and human embryonic kidney HEK-293 cells (CRL-1573, ATCC, Manassas, VA, USA) were cultured at 37 °C under a 5% CO₂ atmosphere in MEM medium, supplemented with 5% to 10% heat-inactivated foetal bovine serum (FBS). A549-Dual™ (A549^DUAL) cells were maintained in growth medium supplemented with nonessential amino acids, 10 µg·mL⁻¹ blasticidin and 100 µg·mL⁻¹ zeocin (InvivoGen, San Diego, CA, USA). Chloroquine phosphate was purchased from Sigma-Aldrich (Saint-Louis, MO, USA). Rat antibody specifically raised against ZIKV E protein Domain III was developed in-house and used in immunoblot with reducing conditions [28 (link)]. Mouse anti-pan flavivirus envelope E protein monoclonal antibody (mAb) 4G2 was purchased from RD Biotech (Besancon, France) and used in immunoblot with nonreducing conditions. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies were purchased from Vector Laboratories (Burlingame, CA, USA).