Figure 5
Adaptation of the assay for an siRNA screening approach. (A) Reduction of relative release efficiency by knock-down of Tsg101. 96-well plates were coated with non-silencing control siRNA or siRNA targeting Tsg101, respectively, as described in methods. 293T cells were seeded onto those plates for reverse transfection and incubated for 18 h at 37°C. Subsequently, cells were transfected with a mixture of pCHIV/pCHIVeYFP or their late domain defective variants, respectively. Supernatants and cell lysates were harvested at 36 hours after this second transfection and relative fluorescence intensities were determined. Relative release efficiencies (RFIsup/RFItotal) were normalized to the mean of the non-silencing control. Mean values and standard deviations from triplicate transfections are shown; black bars and gray bars, respectively, represent results from two independent experiments. (B) Reproducibility of results obtained in a pilot siRNA screen. The graph shows the correlation between normalized eYFP intensities (eYFP intensity of supernatant divided by total eYFP intensity; RFIsup/RFItotal) of two replicates of an RNAi screen investigating host cell functions in HIV-1 assembly and release, comprising a total of 480 wells in 96-well plates. Each dot represents an individual siRNA mediated gene knockdown. The Pearson's correlation coefficient was calculated using GraphPad Prism.