Dihydroartemisinin dha

Two hours post-cytometric quantitation (or 6 h after Percoll synchronization) samples whose stage composition was > 70% rings as determined by flow cytometry were diluted to 2% haematocrit and 0.5% parasitaemia (unless otherwise noted), and 200 μl of culture was aliquoted into 6 wells of a flat bottom 96-well plate. Each treated and untreated sample had three technical replicates: RBC controls were aliquoted into 2 wells at 2% haematocrit and 200 μl. Three wells of parasites and 1 well of the RBC control were treated with 700 nM dihydroartemisinin (DHA) (Sigma-Aldrich); an additional 3 wells of parasites and 1 well of RBC control was treated with 0.02% dimethyl sulfoxide (DMSO) (ThermoFisher) as untreated controls. Parasites were incubated for 6 h, and then washed three times with 150 μl of RPMI to remove drug. Samples were then suspended in CM and placed back in the incubator. Sixty-six hours after drug removal, 20 μl of sample from each well was collected and frozen for qPCR amplification (72 h sample) without any media changes. Plates were then placed back in the incubator for another 48 h, after which 20 μl of sample was again collected and frozen for qPCR amplification (120 h sample).

A final 0.02% gametocytemia was used for the drug assay in order to minimize the cost in parasite culture. To determine drug sensitivity of gametocytes, 100 µl of the cultures from day -1 to 11 were diluted in a complete medium with fresh human erythrocytes to a 2% hematocrit and 0.04% gametocytemia, and placed into each test well of 96-well plates prefilled with the test drugs to a final volume of 200 µl and final hematocrit of 1%. Because young gametocytes at day -1 (stressed schizonts) and day 0 could not be separated, gametocytemias were determined by counting with FCM the fluorescent gametocytes in culture, which contained asexual stage parasites. The plates were incubated at 37°C for 48 h. Chloroquine (CQ), PMQ and dihydroartemisinin (DHA) were purchased from Sigma (St Louis, MO, USA), while pyronaridine (PND) was obtained from Kunming Pharmaceutical Co. (Kunming, Yunnan, China). The stock solutions of CQ (100 mM), PMQ (100 mM), and PND (20 mM) were prepared in RPMI 1640, and DHA (143 mM) was dissolved in DMSO. Two-fold serial dilutions of each drug were made in a complete medium, with the concentration range of each drug shown in Table 1. For each parasite isolate and drug concentration, the assay was performed with at least three biological replicates, each with two technical replicates.

Anti-malarial drugs, pyrimethamine (PYR, MW = 249) and dihydroartemisinin (DHA, MW = 284) were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). DFO (desferrioxamine mesylate or Desferal^®, MW = 657) was from the Novartis Pharma, Basel, Switzerland, Chiang Mai. DFP (GPO-L-One^® or L1, MW = 139) and DFX (Exjade^®, MW = 373) were kindly supplied by the Institute of Research and Development, Government Pharmaceutical Organization, Bangkok, Thailand. SYBR Green I nucleic acid gel-stain (10,000× concentrate in DMSO, Cat. No. S9430) was purchased from Invitrogen, Molecular Probes (Eugene, OR, USA). Calcein acetoxymethyl (CA-AM) was purchased from Invitrogen^® Corporation (CA, USA). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) and epigallocatechin 3-gallate (EGCG, MW = 458) were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). SYTO^® 61 red fluorescent nucleic acid dye (Cat. No. S11343) was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA).

RPMI-1640 (Cat# 11875093) and fetal bovine serum (Cat# 10099-141) were purchased from Gibco (Grand Island, NY, USA). Dihydroartemisinin (DHA) was purchased from Sigma Aldrich (Cat# D7439, St. Louis, MO, USA). Antibodies against KDM3A (Cat# ab106456) was purchased from Abcam (Cambridge, London, UK), anti-p21 (Cat# sc-471) , anti-GAPDH (Cat# sc-25778) and anti-β-Actin (Cat# sc-8432) antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Protease inhibitor cocktail was purchased from KeyGen (Cat# KGP603, Jiangsu, China). BCA protein assay kit and enhanced chemiluminescent substrate kit were obtained from Pierce (Cat# 23225, Thermo Scientific, Rockford, IL, U.S.A). Cell Counting Kit-8 (CCK-8) was purchased from Med Chem Express (Cat# HY-K0301, Shanghai, China).

10058-F4, 10074-G5, JQ1 and dihydroartemisinin (DHA) were purchased from Sigma-Aldrich (St. Louis, MO). The 10058-F4 analogs 12Rh and 28Rh, the 10074-G5 analogs 3JC-91-2 and 3JC91-7 and the Myc proteomimetic compound JKY-2-169 [17 ] were all synthesized and purified as previously described [12 (link), 38 (link)].

Dihydroartemisinin (DHA) was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA), dissolved in DMSO to make a 1000 mM stock solution, and stored at 4°C. The following antibodies were used: β-actin, Cdk4, Cdk6 (Santa Cruz Biotechnology, Carlsbad, CA, USA), VEGF (Abcam Inc., Cambridge, MA, USA), IKKα, MEK1 (Cell Signalling Technology, Inc., Danvers, MA, USA), E2F3, Rac1, E2F1, and CDC42 (Proteintech, Chicago, IL, USA).