Figure 4.

PfVIT and PfE140 are important for P. falciparum growth and may be involved in intracellular iron homeostasis. (A) Representative erythrocytes infected with P. falciparum 3D7 parasites that endogenously express a truncated version of PfVIT or PfZIPCO tagged with GFP (green). (B) Growth rates of knockout parasite lines generated. (C) Representative live 3D7 parasites endogenously expressing PfE140-GFP whose green fluorescence was reduced by glmS-mediated knockdown induced by treatment with 2.5 mM glucosamine for 36 h (GlcN) compared to untreated control (Ctrl). (D) Total parasite fluorescence intensities were quantified as background-corrected integrated densities using ImageJ version 2.9.0/1.53t (Schindelin et al., 2012) and compared using Mann-Whitney test. Images to which no averaging or deconvolution software was applied were used for quantification. (E) The size of the parasites was measured as the area of the region of interest and compared using equal variance unpaired t test. (F) Conditional knockdown of PfE140 induced by treatment with 2.5 mM GlcN results in a growth defect during asexual blood stage development. Live parasites were stained with Hoechst-33342 (blue) and imaged under physiological conditions at 37°C using an SP8 confocal laser-scanning microscope (Leica). DIC, differential interference contrast. Scale bar, 2 µm. Error bars represent 95% confidence intervals of the mean, N the number of parasites analyzed, n the number of independent experiments and Hep treatment with 0.7 µM hepcidin. Growth rates refer to the fold change in parasitemia after two intraerythrocytic developmental cycles in vitro relative to untreated wild-type 3D7 parasites (WT) as determined by flow cytometry with SYBR Green I (Malleret et al., 2011). Statistical significance of growth differences was calculated with two-tailed unpaired t tests with Welch’s correction for unequal variances and adjusted with the Holm-Šídák method for multiple comparisons.