Fig. 2
From: Functional characterisation of components in two Plasmodium falciparum Cullin-RING-Ligase complexes
PfRBX1 and PfSKP1 are essential for intra-erythrocytic P. falciparum survival. (a) Schematic of protein size of wild type, HA-tagged and truncated PfSKP1 in DiCre-PfSKP1-HA transgenic parasite strain. FBOX-binding domain in blue, HA tag in red and Neon Green domain in green colours respectively. (b) Schematic of protein size of wild type, HA-tagged and truncated PfRBX1 in DiCre-PfRBX1-HA transgenic parasite strain. RING domain in yellow, HA tag in red and Neon Green domain in green colours respectively. (c) Immunoblots of schizont stage parental DiCre, DICre-PfSKP1-HA and DiCre-PfRBX1-HA parasites treated with 0 or 10 nM rapamycin for 48 h. DiCre-Rapamycin mediated excision, protein loading, and total ubiquitination of parasites assessed by anti-HA, anti-Aldolase, and anti-Ubiquitin antibodies respectively. Full-length and truncated PfSKP1-HA and PfRBX1-HA is denoted by one and two blue circles respectively. (d) Analysis of ring stage DiCre, DiCre-PfSKP1-HA and DiCre-PfRBX1-HA parasites grown on DMSO or 10 nM Rapamycin for 3 intraerythrocytic cycles. Parasitemia is assessed as a percentage compared to growth of control parasite grown on DMSO. Data are mean ± s.e.m for n = 3 biological replicates with 2 technical duplicates and compared by two-way Anova (Dunnett’s multiple comparison test. (e) Growth of ring stage DiCre, DiCre-PfSKP1-HA and DiCre-PfRBX1-HA parasites on DMSO or 10nM Rapamycin for 1.5 intraerythrocytic cycles. Parasitemia was assessed every 24 h following treatment and assessed as a percentage compared to growth of control parasite grown on DMSO. Data are mean ± s.e.m for n = 3 biological replicates and compared by two-way Anova (Dunnett’s multiple comparison test. (f) Immunofluorescence imaging of DMSO (top panels) and Rapamycin (bottom panels) treated DiCre-PfRBX1-HA schizonts. Parasite membrane integrity and DNA levels visualised using anti-PfGAP45 antibodies and SPY505 DNA dye respectively. (g) Immunofluorescence imaging of DMSO (top panels) and Rapamycin (bottom panels) treated DiCre-PfSKP1-HA schizonts. Parasite membrane integrity and DNA levels visualised using anti-PfGAP45 antibodies and SPY505 DNA dye respectively. (h) Transmission electron microscopy of Rapamycin treated parental DiCre, DiCre-PfRBX1-HA and DiCre-PfSKP1-HA schizont stage parasites. The nuclei and rhoptries have been manually annotated and coloured blue and yellow respectively using a trace feature from Adobe Illustrator that identifies these organelles.
