PmV-GFP-DD clone G6 generation and characterization.

(A) Creation of a PmV-GFP-DD chimera by homologous recombination. The diagram shows the strategy to create C-terminally tagged PmV by integration at the endogenous locus. The plasmid contains sequence from the PmV ORF 3’ end in frame with GFP-DD [24]. Relative positions of BsrGI restriction sites (vertical bars) and the probe are indicated. (B) Southern blot of BsrGI-restricted DNA from the parental strain 3D7, the 3 drug cycles (Selection I, II, III), 3 of the isolated clones, among which G6 is number 3, and the transfected plasmid (at the far right) is shown. Arrows: endogenous gene (blue), and modified PmV locus (green). (C) Live microscopy of the clone expressing the cytosolic YFP-DD chimera (via episomally maintained plasmid) and G6 in presence or absence of 0.5 μM Shield-1. Top to Bottom: bright field (grey), Hoechst (blue), fluorescent proteins (green), Lysotracker (red), merges of the blue and green channels and of all three channels. (D) Western blot of total lysates of parasites at diverse concentrations of Shield-1 (0–1 μM): clone DC6, clone G6 and 3D7 expressing YFP-DD. PmV was detected with both anti-GFP and anti-PmV. BiP detection served as loading control. (E) Live microscopy of the G6 clone expressing the PmV-GFP-DD chimera at various Shield-1 concentrations (0–1 μM). (F) PmV levels from DC6 (green) and G6 (red) were quantified from blot (panel D) and plotted as ratio of PmV to BiP. The last point of the ratio is affected by a reduced BiP expression in 1 μM Shield-1. This is possibly due to the level of toxicity of this molecule at high concentrations. (G) Flow cytometry analysis of the GFP signal of PmV-GFP-DD (clone G6) in presence (green line) or absence (red line) of 0.75 μM Shield-1 in respect of PmV-GFP (clone DC6) (black line). The cellular levels of PmV-GFP(±DD) are similar in the presence 0.75 μM Shield-1 while PmV-GFP-DD is about 4 times less than PmV-GFP in the absence of Shield-1. (H) Growth curve analysis over 10 days for PmV-GFP-DD and DC6 in the presence or absence of 0.75 μM Shield-1. No significant effect on growth was observed in consequence of the reduced PmV levels.