Downregulation of <i>Pf</i>ATM1.

<p>(<b>A</b>) Coomassie-stained SDS-PAGE of purified ~33 kDa <i>PfA</i>TM1-NTE. (<b>B</b>) Strategy for blocking translation of <i>Pf</i>ATM1 using <i>Pf</i>ATM1-Vm. (<b>C</b>) Time-dependent expression of <i>Pf</i>ATM1 in untreated (UT), control morpholino (C-V<sub>m</sub>) or <i>Pf</i>ATM1-V<sub>m</sub> treated parasites at 72 hours (i), 96 hours (ii) and 144 hours (iii) post-treatment. Actin served as loading control. (<b>D</b>) Quantitative analysis of western blots after V<sub>m</sub> treatment. <i>Pf</i>ATM1 levels at 72, 96, and 144 hours post-treatment in untreated and <i>Pf</i>ATM1-V<sub>m</sub> treated cells were normalized with actin and plotted relative to levels in untreated cells (left). Two biological replicates were analysed by two-tailed Student’s t-test; mean and SD are plotted. <i>Pf</i>ATM1 levels from untreated, C-V<sub>m</sub> treated and <i>Pf</i>ATM1-V<sub>m</sub> treated cells were compared at 96 hours post-treatment (right). Mean and SD of three biological replicates are plotted. Significant differences (p <0.05) are in bold. (<b>E</b>) Giemsa-stained parasites representing predominant stages from untreated, C-V<sub>m</sub> and <i>Pf</i>ATM1-V<sub>m</sub> treated sets at different times post-treatment. (<b>F</b>) Parasite stages as % of total parasites in the second and third infection cycle post-treatment. P values (t-test) are shown for comparison of ring stage between control V<sub>m</sub> and <i>Pf</i>ATM1-V<sub>m</sub> at 96 hours, and for late trophozoites between control V<sub>m</sub> and <i>Pf</i>ATM1-V<sub>m</sub> at 108 hours. Significant differences (p <0.05) are highlighted in bold. Parasitemia (%) is given at the top of the bars. (<b>G</b>) Confocal microscopy after staining <i>Pf</i>ATM1-V<sub>m</sub> treated and untreated cells at 96 and 108 hours post-treatment. Cells were stained with DAPI and Mitotracker Red to detect any changes in mitochondrial morphology. Matching parasite stages were selected for comparison. (<b>H</b>) and (<b>I</b>) Western blot of organellar fraction from control untreated (H) and <i>Pf</i>ATM1-V<sub>m</sub>-treated cells (I) probed with antibodies against mitochondrial (<i>Pf</i>IscA2), apicoplast (<i>Pf</i>HU), nuclear (Histone H3) and cytosolic (α-tubulin) proteins. The cytosolic marker protein α-tubulin was not seen in the organelle pellet. Soluble fraction (SF), Pellet (P). (<b>J</b>) and (<b>K</b>) Total iron (J) and glutathione (K) levels in crude organelle fraction of untreated and <i>Pf</i>ATM1-V<sub>m</sub> treated parasites at 108 hours post-treatment. The means and standard deviations from two independent biological replicates are shown. P values (t-test) are indicated.</p> <p>(PDF)</p>