Fig. 8. Knock-down of Fxna expression using siRNAs and shRNAs. (A) Left panel shows that siRNAs decrease Fxna mRNA levels in RK3E cells 48 hours after transfection, as measured by semi-quantitative PCR. Each well was transfected with a different siRNA and the cellular RNA extracted 48 hours later. An 886 bp cDNA fragment was then PCR-amplified using primers annealing to sequences in the coding region of Fxna mRNA. Right panel is a quantitation of the changes detected in the gel shown in the left panel. (B) Left panel is a gel showing that mutating the third base of two codons in the sequence of Fxna mRNA targeted by LV-sh436 rescues Fxna mRNA from siRNA-induced silencing. The mutation creates a silent mutation as it does not change the encoded amino acids. 293T cells were transfected with a plasmid encoding wild-type Fxna mRNA or an Fxna mRNA carrying a silent mutation of the mRNA region targeted by LV-sh436. Six hours later the cells were infected with a lentiviral vector producing LV-sh436 or were left uninfected. The cells were then cultured for 4 days before extracting the RNA for semi-quantitative PCR. Right panel is a quantitation of the changes illustrated in the left panel. Fxna WT, wild-type Fxna mRNA; Fxna-Mut, Mutated Fxna mRNA. Bars are mean±s.e.m. and numbers in parentheses indicate the number of wells per group. ^**, P<0.01 versus both control groups.