Figure 10. Accumulation of positive- and negative-polarity RNA in a recombinant replicon system.

Cultures of HEK293T cells were transfected with SFPQ/PSF-specific (S1) or control siRNAs (siCTRL) as described under Materials and Methods. When the expression of SFPQ/PSF was known to be down-regulated, the cultures were further transfected with plasmids expressing the polymerase subunits and the NP, as well as with a genomic plasmid expressing a pseudoviral gene containing the cat gene in negative polarity. As control, the cultures were transfected with empty plasmids. At 24 hours post plasmid transfection total cell extracts were prepared and total cell RNA was isolated. Significance was determined by the Student's t test. (A) The accumulation of CAT protein is indicated as percent of maximal values. (B) The accumulation of positive-polarity RNA was determined by hybridisation with CAT-specific probes and is presented as percent of maximal values. (C) The accumulation of negative-polarity RNA was determined by hybridisation with CAT-specific probes and is presented as percent of maximal values. The values presented represent the average and standard deviation of 3 independent transfection experiments. (D) The silencing of SFPQ was controlled by Western-blot, using actin as loading control. The level of expression of PA and NP was ascertained by Western-blot.