Lipid internalisation assays were performed according to the methods of Yabas et al. [64] with some modifications similar to Arashiki et al. [23]. A culture containing uRBCs and 5–10% late-stage (trophozoite/schizont) iRBCs was washed twice in PBS with 10 mM D-Glucose (PBS-G). RBCs were pre-treated with 100 μM Furosemide (Sigma) or solvent control (0.06% v/v methanol) for 5 minutes, and then 0.5 mM sodium orthovanadate (Sigma) or the corresponding volume of buffer only, and incubated for 5 minutes.

All lipids were sourced from Avanti Polar Lipids. 5 μM of NBD-PS (1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphoserine), NBD-PE (1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphoethanolamine), NBD-PC (1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine)), or a corresponding volume of solvent-only control (0.5% v/v ethanol) was added to triplicate wells of treated/untreated RBCs and incubated at room temperature in the dark, to allow NBD-lipids to translocate to the inner membrane leaflet. After 20 minutes, cells were washed twice with ice-cold 4% w/v lipid-free Bovine Serum Albumin (Sigma), to extract NBD-lipids remaining in the outer membrane leaflet. Cells were resuspended in PBS-G with 5 μg/mL Hoechst 33342 and incubated for 20 minutes on ice. Images and flow cytometry data were collected and processed as described above. The ATP-dependent fraction of lipid internalisation was calculated from the difference between total (untreated) and ATP-independent (vanadate-treated) lipid internalisation. Data were normalised between experiments (untreated uRBC set to 1) and analysed as specified. For figures comparing mean fluorescence intensity without normalisation (Figs Figs1D1D and S1B), data were fitted with a Linear Fixed Effects Model to calculate the Least Squared Means in RStudio, with date of experiment as a blocking factor to minimise the effect of fluctuations in machine settings [103]. Significance tests were computed by the ‘Difference of Least Squared Means’ function on this model.

The lipid internalisation assay was repeated on parasites isolated from their host RBCs by treatment with 0.15% w/v saponin (Sigma), after magnetic enrichment of iRBCs using a SuperMACS II Magnet (Miltenyi Biotec). The experiment was performed in malaria saline (135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 20 mM glucose, 25 mM HEPES, pH. 7.1) instead of PBS-G. Images and flow cytometry data were collected and processed as described above. Data were normalised to the highest fluorescence value (untreated NBD-PC internalisation), and analysed in Prism 8 as specified.

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