Figure 2

PAI-1 expression levels in WRN knocked down fibroblasts. Five synchronized fibroblast cultures presenting heterozygosis for WRN (1367CR/1074LF) were transfected with a molecule of iRNA against WRN messenger (iRNA; panel D), or with a scrambled control RNA (Scrambled; panel C). PAI-1 and WRN expression was evaluated by semiquantitative RT-PCR at several times post-transfection. Normalized values of mRNA were grouped as mean ± standard error of arbitrary units. ANOVA was used to test differences across treatments (control, scrambled and iRNA). * Significant differences compared with scrambled by Tukey test; P < 0.01. Cells cultures were subjected to immunoblotting with anti-WRN antibody to monitor WRN knocking down (panel A). Semiquantitative RT-PCR was done at 26th cycle of amplification from total RNA isolated from cell cultures, products were separated by electrophoresis in 2% agarose gel and stained with ethidium bromide. The intensity of the fluorescence was measured and integrated by "Gel Doc" software. All RT-PCR experiments were done by triplicated (panel B) M = molecular weight ladder.