Figure 9

Influences of endogenous 16.4.1 proteins on transactivation capacity of Rev in human cells. (A and B) Downregulation of 16.4.1-GFP expression in HeLa cells by siRNAs. HeLa cells stably expressing 16.4.1-GFP were transfected with a pre-synthesized siRNA directed against a 16.4.1 specific sequence (siRNA-16.4.1) or a non-specific siRNA (siRNA-nsp). Expression of 16.4.1-GFP was quantified by flow cytometry in 40.000 cells. Panel A shows an overlay of GFP-expressing cell populations in untransfected cells (green curve) and after transfection with the 16.4.1-specific siRNA (blue) and the non-specific (red). 16.4.1-GFP expression in untransfected cells was set at 100% and the percentage of down regulation of green fluorescence indicated. siRNA-16.4.1 reduced expression levels of GFP-tagged 16.4.1 to 36% which was similar to the silencing effect of si-GFP which was analyzed as control (not shown). Higher levels of 16.4.1-GFP expression (81%) were observed in cells transfected with the unspecific siRNA-nsp. (C) Enhancement of Rev transactivation capacity by siRNAs targeted against 16.4.1. 293T cells were transfected with siRNA-16.4.1 or siRNA-nsp and Rev activities compared with mock-transfected cells (i.e. cells treated with the RNAi transfection reagent lacking RNA). Transfection of siRNA-16.4.1 increased Rev activity by 17% whereas the unspecific siRNA-nsp increased Rev activity by only 1%.