Statistical significance was determined using a Brown-Forsythe and Welch ANOVA ( A, B) or a two-way ANOVA with TukeyвЂ™s test for post hoc analysis ( CвЂ“F). Data in ( CвЂ“F) represent three to four biological replicates conducted in technical triplicate. Data in ( B) represent two to four biological replicates conducted in technical triplicate. Data in ( A) represent four to seven biological replicates conducted in technical triplicate. Average PV number per field ( C) and PV size ( D) were quantitated for 36 hr infections while total area infected per sample ( E) and average foci size ( F) were quantitated for 96 hr infections. Cells were fixed, stained with anti-GFP and anti-RFP antibodies, and imaged using a Cytation3 Imager. ( CвЂ“F) WT A549 cells were transduced with TRIP.RARRES3 or TRIP.FLUC control and infected 72 hr later with CTG-GFP for 36 ( C, D) or 96 ( E, F) hr. Average cell number ( A) and the percentage of SYTOX staining cells ( B) were determined. As a positive control, cells were permeabilized by treatment with methanol (MeOH) for 5 min prior to staining. After 60 hr, cells were stained with Hoechst 33342, SYTOX green, and imaged with a Cytation3 Imager. ( A, B) Wild-type (WT) A549 cells were either not transduced (NT) or transduced with TRIP.RARRES3 or TRIP.FLUC control and split 48 hr later. ISGs enhancing or restricting infection >20% relative to control with p0.05, **p<0.01, ****p<0.0001. Statistical significance was determined using a two-way ANOVA with TukeyвЂ™s test for post hoc analysis. After 72 hr, cells were infected with CTG-GFP for 36 hr, fixed, stained with anti-GFP and anti-RFP antibodies, and imaged with a Cytation3 Imager. ( E) A549 cells were transduced with a lentiviral expression cassette co-transcriptionally expressing tagRFP and an ISG of interest in a one gene per well format. ( D) Illustration of the method used to conduct the screen presented in ( E). ( AвЂ“C) Average parasitophorous vacuole (PV) size ( A), PVs per field ( B), and the percentage of vacuoles containing в‰Ґ8 parasites ( C) was quantitated for data from 36 hr image sets. ( AвЂ“C) Cells were fixed, stained with anti-GFP and anti-RFP antibodies, and imaged using a Cytation3 Imager. A549 cells were treated with indicated concentrations of IFNОі for 24 hr and subsequently infected with the type III strain CTG expressing GFP (CTG-GFP) for 36 hr.

Cellprofiler 3.1.9