Figure 3

DFMO arrest and putrescine reversal associated to G1/S cell cycle control in intraerythrocytic P. falciparum parasites. In all instances, ■ refers to control parasites, ▼ refers to DFMO-treated parasites, and ● refers to putrescine-reversed parasites. (a) DFMO dose-response curves of asexual P. falciparum 3D7 proliferation over 96 h (initiated with ring-stage parasites, 1% haematocrit, 1% parasitaemia) at 37 °C in the absence or presence of 2 mM putrescine. Proliferation is expressed relative to untreated controls, with data averaged from n = 6 biological replicates and shown ± S.E. (b) Synchronised P. falciparum 3D7 cultures were treated with DFMO alone (IC₉₀) or with putrescine (2 mM, after 24 h DFMO pressure) and parasitaemia monitored over 96 h with SYBR Green I fluorescence (10 000 infected erythrocytes counted). *P < 0.05, student t-test. Data from n = 4 biological replicates in duplicate (c) Gametocytaemia of P. falciparum NF54 cultures over 14 days, determined microscopically. Control or DFMO-treated parasites (IC₉₀, 24 h treatment of ring-stage parasites before DFMO removal with fresh media) before gametocytogenesis induction. Data averaged ± S.E. from n ≥ 3 biological replicates with >1000 cells counted; where not shown, error bars fall within symbols. (d) Flow cytometric analysis of nuclear division in P. falciparum parasites following life cycle arrest and subsequent re-entry into the life cycle. Parasites (1% haematocrit, 10% parasitaemia) were sampled after 12 hpi, 36 hpi, 39 hpi, 42 hpi or treated with DFMO (IC₉₀) for 24, 27 or 30 h before sampling. Additionally, following 24 h of DFMO treatment, putrescine (2 mM) was added to stimulate cell cycle re-entry, and samples taken 3, 6, 12 h after reversal. The nucleic acid content of parasitised erythrocytes was determined by consecutive staining with both SYBR Green I (DNA fluorescence) and PyroninY (RNA fluorescence); detected in the FITC or PE channel, respectively. Overlaid histograms for a representative sample of biological triplicates. DNA content (SYBR Green I fluorescence) confirmed using fluorescent microscopy on a Zeiss LSM 880 Confocal Laser Scanning Microscope.