Figure 2. Confirmation of gene silencing in a screening format and overview of the screening procedure.

A. Eighteen target genes represented in the library were silenced by siRNA transfection of HeLa cells in 96 well plates. The percent decrease in mRNA levels of target genes was determined using qRT-PCR 72 h post-transfection. n = 2 independent experiments, each with triplicate samples. Error bars, SEM. B. Schematic flowchart showing the siRNA screening strategy for identification of host factors playing a role in Toxoplasma invasion. The strategy consisted of three main steps of transfection, invasion and measurement. Host cell viability was assessed using CellTiter-Fluor™, which measures protease conversion of a quenched membrane permeable peptide substrate (glycine-phenylalanine-7-Amino-4-trifluormethylcoumarin). Parasite invasion was assessed using the luciferin-based reagent Steadylite Plus™, which measures the inducible luciferase activity of U-luc parasites.