Figure 4

siRNA-mediated double knockdown of USF1 and USF2 reduces P1.1 activity. A) To examine the transcript specificity of knockdowns, Jeg3 trophoblast cells were transfected with pools of siRNAs targeting USF1 (siUSF1), USF2 (siUSF2) or a siControl pool, which does not target any known transcript. Mock-transfected cells treated only with the transfection vehicle were used to normalise measurements. USF transcripts were quantified by real-time PCR 24 h after transfection. The relative transcript abundance was calculated by dividing measurements from siRNA treated cells with measurements from mock-transfected cells. Mean values ± S.E.M. of n = 3 experiments are shown. B) To analyse the effect of USF knockdown on P1.1-dependent transcription, Jeg3 cells were transiently transfected with the P1.1-luciferase reporter gene plasmid along with siRNAs (siUSF1, siUSF2 or siUSF1+USF2). Jeg3 cells transfected with a non-targeting siRNA (siControl), or without siRNA served as controls. Luciferase activities were measured 72 h after transfection. Results (relative light units, rlu) are shown as means ± S.E.M. of at least n = 3 experiments. Only the double knockdown resulted in a significant reduction of the reporter gene activity (p < 0.05), as indicated by an asterisk.