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6 protocols using artemisinin

1

Antimalarial Compound Screening Protocol

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Unless otherwise stated all reagents were from Sigma-Aldrich (St. Louis, MO). DCM was from ThermoFisher (Waltham, MA, USA); artemisinin was from Cayman Chemical (Ann Arbor, MI, USA); artemether, artesunate, and dihydroartemisinin were gifts from Prof. J. Plaizier-Vercammen (Brussels, Belgium); deoxyartemisinin was from Toronto Research Chemicals (North York, ON, Canada); amodiaquine HCl hydrate (Cat #: 562290) and imanitib (Cat # 100956) were from Medkoo Biosciences Inc. (Morrisville, NC, USA); EMEM (Cat # 30–2003) and XTT reagent (Cat # 30–1011k) were from ATCC; PrestoBlue was from Life Technologies (Cat #P50201); Renilla-Glo was from Promega (E2720).
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2

Antimalarial Compound Screening Protocol

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Unless otherwise stated all reagents were from Sigma-Aldrich (St. Louis, MO). DCM was from ThermoFisher (Waltham, MA, USA); artemisinin was from Cayman Chemical (Ann Arbor, MI, USA); artemether, artesunate, and dihydroartemisinin were gifts from Prof. J. Plaizier-Vercammen (Brussels, Belgium); deoxyartemisinin was from Toronto Research Chemicals (North York, ON, Canada); amodiaquine HCl hydrate (Cat #: 562290) and imanitib (Cat # 100956) were from Medkoo Biosciences Inc. (Morrisville, NC, USA); EMEM (Cat # 30–2003) and XTT reagent (Cat # 30–1011k) were from ATCC; PrestoBlue was from Life Technologies (Cat #P50201); Renilla-Glo was from Promega (E2720).
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3

Phagocytosis Assay with Zymosan

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We obtained the following reagents from the indicated sources/vendors: pHrodo® Red Zymosan Bioparticles® Conjugate (Thermo Scientific; P35364, 1 mg/ml PBS stock), pHrodo® Red AM (Thermo Scientific; P35372, 10 mM DMSO stock), natural drug library (Selleckchem; L1400, 10 mM DMSO stock, see Supplementary Table 1 for drugs tested), LC3 detection dye (Dojindo Molecular Technologies, Inc.; D675-10), ROS/RNS kit (ENZO Life Science; ENZ-51001-200), artesunate (Cayman Chemicals; 11817, 30 mM DMSO stock), artemisinin (Cayman Chemicals; 11816, 30 mM DMSO stock), dihydroartemisinin (Abcam; ab142690, 30 mM DMSO stock), and clodronate liposomes (Liposoma B.V.; C-005). NPD1 was gifted from Resolvyx Pharmaceuticals. All reagents were used in the dose and manner described in the figure legends.
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4

In Vitro Cytokine and CYP450 Assays

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Chemicals and reagents were from Sigma Aldrich (St. Louis, MO, USA) unless otherwise stated. Artemisinin was from Cayman Chemical (Ann Arbor, MI, USA); deoxyArtemisinin from Toronto Research Chemicals (North York, ON, Canada). Lipopolysaccharide was Escherichia coli serotype O111:B4 and dissolved in sterile water at 1 mg/mL before storage at −20 °C. LEGEND MAXTM Enzyme-linked immunosorbent assay (ELISA) kits for rat TNF-α and IL-6 were from BioLegend (San Diego, CA, USA). ELISA kits for rat IL-10 were from Thermo Fisher Scientific (Waltham, MA, USA). P450-Glo CYP2B6 and CYP3A4 kits were from Promega (Madison, WI, USA). Human liver microsomes (HLMs) from a 200-donor pool of male and female donors were from Sekisui XenoTech (Kansas City, KS, USA).
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5

Artemisinin Compound Treatments in Fibroblasts

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Drug treatments were performed as follows. A. annua tea was diluted in a growth media to 50 µM ART; A. afra tea was diluted with water to 1/2, 1/4, or 1/8 strength of the A. annua tea on a dry-weight basis as needed; artemisinin (ART; Cayman Chemical, Ann Arbor, MI, USA, 11816), artesunate (AS; Cayman Chemical 11817), artemether (AM; gift from Prof. J. Plaizier-Vercammen (Brussels, Belgium)), dihydroartemisinin (DHA; Cayman Chemical 19846), and deoxyartemisinin (dART; Toronto Research Chemicals, Toronto, Ontario, Canada, D232150) were dissolved in DMSO as 1000× stock solutions, filter sterilized, and added to a final concentration of 50 μM. DMSO and water were the solvent controls for the artemisinic compounds and teas, respectively. Fibroblasts (passages 10–14) were seeded in 6-well plates in 2 mL medium at a density of 6250 cells/cm2. After overnight attachment, media were aspirated and replaced with test drugs in growth media at the indicated concentrations and incubated at 37 °C and 5% CO2 in high humidity. After 4 d of drug treatment, cells were harvested, washed with 1× Dulbecco’s phosphate-buffered saline (DPBS; Thermo Fisher Scientific 14190136), snap frozen in liquid nitrogen, and stored at −80 °C until the time of RNA extraction for analysis.
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6

Antimalarial Drug Combination Screening

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Unless otherwise stated all reagents were from Sigma-Aldrich (St. Louis, MO, USA). DCM was from ThermoFisher (Waltham, MA, USA); artemisinin was from Cayman Chemical (Ann Arbor, MI, USA); artemether, artesunate, and dihydroartemisinin were gifts from Prof. J. Plaizier-Vercammen (Brussels, Belgium); deoxyartemisinin was from Toronto Research Chemicals (North York, ON, Canada); amodiaquine HCl hydrate (Cat #: 562290) and imatinib (Cat # 100956) were from Medkoo Biosciences Inc. (Morrisville, NC, USA); EMEM (Cat # 30–2003) and XTT reagent (Cat # 30-1011k) were from ATCC; PrestoBlue was from Life Technologies (Cat #P50201); Renilla-Glo was from Promega (E2720).
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