(A) Synchronous cycle 2 parasites of the indicated clone (parasitaemia ~5%) treated 72 hr previously with DMSO or rapamycin in cycle 1 were suspended in osmotic lysis buffer containing 280 mM sorbitol or in PBS, and the resulting cell lysis determined by measuring the absorbance of the supernatant at 405 nm. An equal volume of parasite culture was lysed in 0.15% (w/v) saponin to give a value for 100% lysis and all other absorbance values normalized to this. Data were averaged from three biological replicate experiments. Statistical significance was determined by a two-tailed t-test; significance levels are indicated: p≤0.001, ***; p≤0.01, **;p≤0.05, *; and p>0.05, non-significant (ns). (B) Uptake of 5-ALA by erythrocytes infected with either DMSO-treated or rapamycin-treated rhopH3-loxP clone 5F5 parasites at cycle 2. Cultures were incubated overnight with 200 μM 5-ALA and uptake of the compound and its subsequent conversation to PPIX in infected erythrocytes visualized by fluorescence microscopy. Infected erythrocytes were visualized by staining with Hoechst 33342. Top panels show fields of view containing multiple infected erythrocytes of the indicated strain. Scale bar, 50 μm. Bottom panels show individual infected erythrocytes. Scale bar, 5 μm. (C) Quantitation of the levels of uptake of 5-ALA by infected erythrocytes. For each condition, a total of 1300 Hoechst-positive cells were analyzed for intensity of PPIX fluorescence using MetaMorph (Molecular Devices) and a statistical significance was determined by a two-tailed t-test. Significance levels are indicated: p≤0.0001, **** and p>0.05, non-significant (ns). (D) Flow cytometry analysis of 5-ALA-treated parasites. Uptake of 5-ALA and its subsequent conversation to PPIX in cycle 2 parasites following treatment in cycle 1 with rapamycin or DMSO was determined by flow cytometry of Hoechst stained parasites. Gating was applied to distinguish Hoechst negative cells (red population), Hoechst positive/PPIX negative cells (green population) and Hoechst positive/PPIX-positive cells (purple population). For the 1G5DC parental and RhopH3-loxP NE parasite clones, most of the parasites were positive for both Hoechst and PPIX fluorescence regardless of their treatment with rapamycin or DMSO. In contrast, for rapamycin-treated rhopH3-loxP clones 5F5 and 4B11, most of the parasites were Hoechst positive/PPIX negative indicating a defect in 5-ALA uptake.