Figure 3

HIV-1 Tat and Rev can suppress Dicer dependent RNAi process involving ARM. HEK 293 T cells were transfected with an expression plasmid for HIV-1 Tat, Rev and their corresponding ARM deletion mutants i.e. ∆Tat and ∆ Rev (1 μg each). In ARM mutants the ARM region was deleted (48-60 in Tat and 35-50 in Rev). (A) shows the expression levels of wild type and ARM mutants of Tat and Rev respectively. The stability of Tat, Rev and their corresponding ARM deletion mutants was determined using Cycloheximide chase for 8 hours (B). HEK 293 T cells were co-transfected with expression plasmid for GFP, Dicer dependent shRNA against GFP (0.5 μg) along with Tat, Rev and their corresponding ARM deletion mutants. GFP expression was measured at 48 hours post transfection. (C) shows fluorescence microscopic images of RNAi suppression by HIV-1 Tat, Rev and their ARM deletion mutants. (D) is a Flow cytometric analysis to show the suppressor effect of HIV-1 Tat, Rev and ARM deletion mutants on GFP expression in Dicer dependent RNAi suppressor assay. Dot plot depicts the number of cells (counts) on Y-axis versus the expression of GFP reporter (FL1) on X-axis. (E) is a histogram plot of suppressor assay. The p values indicate that the difference between the wild type Tat/Rev and corresponding ARM mutant is significant.