PfMF3 localizes to the parasite mitochondrion. (a) Map of plasmid used for episomal overexpression of GFP-tagged pf mfr3. An empty vector (no pf mfr3 insert) was used for transfection of a control (Dd2_empty) parasite line. (b) PCR amplification of the blasticidin-resistance(BSD Deaminase) marker in both parasite lines confirms the successful transfection of both control and overexpression/ tagging parasite lines, while PCR amplification of the chimeric MFR3-GFP template confirms the presence of the MFR3-GFP episome in the overexpression line(Dd2_mfr3over) but not in the control (Dd2_empty). (c) The sensitivity of parasites overexpressing pf mfr3 (Dd2-MFR3_over) and the corresponding control line (Dd2_empty) was evaluated against MMV085203, GNF-Pf-3600, and other antimalarial compounds with known mechanisms of action. Bars represent mean ± SD IC 50 values from at least three independent biological replicates. Pairwise comparisons between parasite lines were performed using the Student’s t test. Additional data can be found in Table 9. (d) Blood-stage parasites expressing a GFP-tagged version of PfMFR3 were costained with MitoTracker Red (200 nM) and DAPI and then imaged using confocal microscopy. The blue signal pertains to the DAPI-stained parasite nuclei; the red signal represents the parasite mitochondrion, and the green signal, GFP-tagged PfMFR3. Rocamora F, Gupta P, Istvan ES, Luth MR, Carpenter EF, Kümpornsin K, Sasaki E, Calla J, Mittal N, Carolino K, Owen E, Llinás M, Ottilie S, Goldberg DE, Lee MCS, Winzeler EA. PfMFR3: A Multidrug-Resistant Modulator in Plasmodium falciparum. ACS Infect Dis. 2021 7(4):811-825. PMID: 33715347