Figure 2. A kinome-wide RNAi screen identifies host genes that influence P. berghei sporozoite infection of Huh7 cells.

(A) Schematic illustration of the three screening passes with increasing stringency criteria. (B) Plot of pass 1 of the RNAi screen representing the effect of 2181 siRNAs targeting 727 human genes on Huh7 cell infection by P. berghei sporozoites and cell nuclei count. Infection rates for each experimental condition were normalized against cell confluency. The horizontal lines represent 100%±2.0 s.d. of the average of all infection data in the assay. Each circle represents one siRNA (mean of triplicate values). Negative controls appear as blue and green circles. corresponding to untreated cells and cells transfected with a non-specific control siRNA. respectively. Red circles highlight the siRNAs targeting the 73 candidate genes selected to undergo a second screening pass. The shaded areas correspond to cell numbers outside the ±40% interval centred on the average number of nuclei for the whole dataset. (C) Plot of 2 independent runs of pass 2 of the RNAi screen representing the effect of 227 siRNAs targeting 73 human genes on Huh7 cell infection by P. berghei sporozoites and cell nuclei count. Shading and colour attributions are the same as in panel (B). with red circles representing the siRNAs targeting the 16 genes selected to undergo a third screening pass. The horizontal lines represent 100%±2.0 s.d. of the average of all the negative controls in the assay. (D) Plot comparison of the 2 runs of pass 2 of the RNAi screen. Colour attributions are the same as in panels (B. C). The comparison reveals a high correlation (R = 0.88) between the duplicate runs of pass 2 of the screen (diagonal line). The horizontal and vertical lines represent 100%±2.0 s.d. of the average of all the negative controls in the assay. (E) Plot of pass 3 of the RNAi screen representing the effect of 37 siRNAs targeting 16 human genes on Huh7 cell infection by P. berghei sporozoites and cell nuclei count. Remaining mRNA levels following RNAi were determined for each of these genes by qRT-PCR (see text and Figure 2F). Colour attributions and shading are the same as in (B. C. D). Red circles highlight siRNAs targeting the genes for which at least two independent siRNAs led to an infection increase or decrease above or below ±3.0 s.d. of the average of all the negative controls in the assay. respectively. The horizontal lines represent 100%±3.0 s.d. of the average of all the negative controls in the assay. (F) Effect of siRNA on infection rates versus remaining mRNA levels for the 7 genes targeted by the siRNAs highlighted in red in (E). Each circle represents one siRNA (mean of triplicate values). For all genes except GUK1 and HCK. represented in light grey. a positive correlation between infection rate and remaining gene-specific mRNA levels is observed. Shading attributions are the same as in (B. C. E). The horizontal lines represent the same as in E (100%±3.0 s.d. of the average of all the negative controls in the assay). The axes on the bottom left of the panel denote the scale of each of the plots in the panel.