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. 2018 Feb 20;9(1):e01800-17. doi: 10.1128/mBio.01800-17

FIG 1 .

FIG 1 

RNAi-based assay to identify host factors required for intracellular survival of Y. pestis. To determine whether reproducible RNAi could be achieved in RAW264.7 macrophages, cells were reverse transfected with siRNAs targeting indicated genes. (A and B) Forty-eight hours posttransfection, cells (n = 3) were harvested for RNA isolation and qRT-PCR (data represent the level of gene expression compared to the level for the scrambled siRNA control) (A) or protein isolation for Western blot analysis (β-actin was used as a loading control) (B). α-GAPDH, anti-GAPDH antibody; α-βactin, anti-βactin antibody. To demonstrate that the Y. pestis CO92 pCD1(-) LuxPtolC bioreporter accurately represents intracellular bacterial numbers, RAW264.7 macrophages were infected with Y. pestis CO92 pCD1(-) LuxPtolC at the indicated MOIs (n = 12), and extracellular bacteria were killed with gentamicin. (C) Bioluminescence (in relative light units [RLU]) of intracellular bacteria was determined at 1, 4, 8, and 18 h postinfection. (D) At 18 h, cells from each MOI (n = 3) were lysed, and bacterial numbers (CFU) were determined and compared to 18-h bioluminescence (in RLU). (E) To demonstrate that RNAi targeting specific genes could impact the intracellular survival of Y. pestis, RAW264.7 macrophages were transfected with siRNAs targeting Rab2A or COPβ1. Forty-eight hours posttransfection, macrophages were infected with Y. pestis CO92 pCD1(-) LuxPtolC (MOI of 10). Extracellular bacteria were killed with gentamicin, and intracellular bacterial bioluminescence was monitored over time. Data are represented as percent RLU of scrambled (Scr) siRNA control. (F) To demonstrate the robustness of the assay, RAW264.7 macrophages (n = 48) were reverse transfected with either scrambled siRNA (negative control) or siRNA targeting Copβ1 (positive control). Forty-eight hours posttransfection, macrophages were infected with Y. pestis CO92 pCD1(-) LuxPtolC (MOI of 10). Extracellular bacteria were killed with gentamicin, and intracellular bacterial bioluminescence was determined at 2 and 10 h postinfection. The Z’ factors from four independent experiments are shown (the bars are means). (G) Overview of optimized high-throughput assay for RNAi screening.