Fig 1. NBD-lipid internalisation at the red blood cell (RBC) membrane differs between uninfected (uRBCs) and infected (iRBCs) cells.

(A) Subcellular localisation of NBD-lipids after internalisation, visualised by deconvolution fluorescence microscopy. NBD-lipid fluorescence was detected at 475 nm (ex)/ 525 nm (em) and Hoechst fluorescence (parasite DNA) was detected at 390 nm (ex)/ 435 nm (em). Shown are representative images for uninfected (top) and infected (bottom) RBCs incubated with NBD-PS (upper rows), NBD-PE (centre rows) and NBD-PC (lower rows) after extraction of NBD-lipids remaining in the outer layer. Scale bar = 4 μm. (B) NBD-lipid internalisation, measured by fold-change of NBD mean fluorescence intensity (MFI) of whole cells in flow cytometry after extraction of NBD-lipids remaining in the outer layer. Cells were treated with 0.5 mM vanadate in calcium-free media to measure only the ATP-independent portion of internalisation. Shown are percentage of mean values (± S.D.) NS = not significant; ** = p < 0.01; *** = p < 0.001 (ANOVA). n = 3 independent experiments. (C) ATP-dependent fraction of NBD-PS internalisation in RBCs, calculated from the difference between total (untreated) and ATP-independent (vanadate treated) fractions (see Methods). Shown is normalised mean fluorescence intensity (± S.D.), *** = p < 0.001 (Mann-Whitney Test). n = 3 independent experiments. (D) NBD-lipid internalisation with treatment of 100 μM furosemide, measured by mean fluorescence intensity (MFI). Units are arbitrary. Shown are Least Square Means (± 95% Confidence Interval). NS = not significant (Difference of Least Square Means test). n = 3 independent experiments.