(A) Western blot showing regulation of the PfNCR1apt by aTc. Trophozoite-stage parasites were harvested from the replication cycle in which aTc was removed (cycle 0), as well as the following two cycles. PfNCR1 was detected using a C-terminal HA-tag. The ER membrane protein plasmepsin V (PM-V) was used as a loading control. Note that the two bands recognized by α-PM-V antibody correspond to the full-length protein and a proteolytic fragment of the protein produced during the membrane isolation. Expected sizes: 171 k Da for PfNCR1-HA, 69 k Da for PM-V. This experiment was done two times. (B) Replication of PfNCR1apt parasites. Using a flow cytometry assay, the replication of two PfNCR1apt clones was monitored over two weeks. +aTc is in black and solid lines, -aTc is in red and dashed lines. Cultures were seeded at 1% parasitemia, and subjected to daily media changes, and/or sub-culturing. Cumulative parasitemias were calculated by multiplying with dilution factors. One representative experiment with technical triplicates is shown. The inset magnifies the initial time points. Doubling times in days are as follows (95% confidence intervals in parentheses): clone 1 -aTc = 1.596 (1.546–1.650), R2 = 0.9964; clone 1 +aTc = 0.8663 (0.8218–0.9159), R2 = 0.9930; clone 2 -aTc = 1.463 (1.417–1.512), R2 = 0.9965; clone 2 +aTc = 0.7776 (0.7559–0.8005), R2 = 0.9981. This experiment was done four times. (C) Complementation of PfNCR1apt rescues growth phenotype. Wild-type PfNCR1 was stably expressed in the PfNCR1apt background. Replication of parasites was monitored over two weeks. +aTc is in black and solid line, -aTc is in red and dashed line. One representative experiment with technical triplicates is shown. Doubling times in days are as follows (95% confidence intervals in parentheses): -aTc = 1.152 (1.036–1.298), R2 = 0.97; +aTc = 1.166 (1.039–1.329), R2 = 0.96. This experiment was done four times. Note that the complemented strain grows less well than PfNCR1apt with aTc (B), but that there is no significant difference ±aTc. (D) Expression level of PfNCR1 correlates with sensitivity to MMV009108. Concentration response curves using a flow cytometry-based growth assay. After aTc washout, aTc was replenished in triplicate cultures at different concentrations and parasitemias were measured after 72 hr. aTc concentrations are indicated. This experiment was done three times. (E–H) PfNCR1 K/D hypersensitizes parasites to three compounds. Concentration-responses of PfNCR1apt parasites to E) MMV009108, (F) MMV028038, (G) MMV019662, and H) mefloquine (MFQ) (control compound) without aTc (red open symbols, dashed lines) or with 500 nM aTc (black symbols, solid lines) after 72 hr. One representative experiment with technical triplicates is shown. The experiment in E) was done three times the experiments in F-H) were done two times.