Janus platform

Serial dilutions of aTc, atovaquone, quinine, and bafilomycin A were generated to yield final concentrations ranging from 40–0.462 nM, 25–0.048 nM, 432–0.84 nM, and 40–0.08 nM, respectively. Synchronous ring-stage PfRAP01 and PfRAP21 conditional knockdown lines as well as a control cell line expressing an aptamer-regulatable fluorescent protein were maintained in high aTc (500 nM), low aTc in the case of PfRAP01 (8 nM) and PfRAP21 (5 nM) or no aTc, and were distributed into 384-well assay plates (Corning^®, 89176-442). Compounds were transferred to the parasite-containing plates using the Janus^® platform (PerkinElmer). DMSO- and dihydroartemisinin-treatment (500 nM) served as reference controls. Growth inhibition was analyzed after 72 and 120 h using the Renilla-Glo(R) Luciferase Assay System (Promega, E2750) and the GloMax® Discover Multimode Microplate Reader (Promega). IC₅₀ values were obtained from corrected dose-response curves and plotted using GraphPad Prism 8.

Stock solutions of compounds were serially diluted to yield final in-assay concentrations ranging from 0.005-2.8 μM for harzianin NPDG H and alamethicin, 0.0006-0.312 μM for harzianin NPDG I, 0.008-4 μM for trichorzin HA II, and 0.05-0.00009 μM for mefloquine (positive control). As a control cell line, a parasite line expressing an aptamer-regulatable fluorescent protein integrated in the cg6 locus⁸⁴ (link) was assayed in parallel. Synchronous ring-stage parasites in the presence (0.5 μM) and absence of aTc were dispensed into 384-well polystyrene microplates (Corning®), and compounds added using the Janus® platform (PerkinElmer). DMSO- and DHA (0.5 μM) served as normalization controls for maximal and minimum signal. Luminescence was measured after 72 h and IC₅₀ values were obtained from normalized dose-response curves using GraphPad Prism Version 8.