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. 2013 May 31;8(5):e64693. doi: 10.1371/journal.pone.0064693

Figure 1. Optimization of target protein knockdown and Toxoplasma invasion assay for high-throughput RNAi screen.

Figure 1

A. HeLa-GFP cells were transfected with non-targeting (NT) siRNA or siRNA targeting GFP by reverse transfection and examined by immunofluorescence 48 h and 72 h post-transfection. B. HeLa-GFP cells were mock-transfected or transfected with non-targeting siRNA or siRNA against GFP and analyzed by immunoblotting 72 h post-tranfection using anti-GFP or anti-actin antibodies. C. HeLa cells were reverse-transfected with either non-targeting siRNA (lysed cells, NTsiRNA or tachyplegin) or PLK1 in 384-wells. 48 h post-transfection, a set of HeLa cells transfected with NT siRNA were treated with passive lysis buffer to lyse cells. 72 h post-transfection, freshly harvested U-Luc parasites untreated or treated with tachyplegin (chemical inhibitor of invasion) were added onto host cells and incubated at 37°C for 3 h. Steadylite Plus™ was added to the plate and luciferase activity was measured using PHERstar system. n = 3 independent experiments each with triplicate samples. Error bars, SEM. D. Host cell viability was assessed by adding CellTiter-Fluor™ and the values obtained with cells transfected with NT siRNA only were set at 100%. n = 3 independent experiments each with triplicate samples. Error bars, SEM.