Figure 4

Decrease in steady state mRNA levels is due to altered rates of messenger degradation which is not ARE dependent. A) Decay of pGL4.71P-C2 mRNA (black squares) compared with that of pGL4.71P control (black diamonds). SK-N-BE cells were transiently transfected with reporter gene constructs and the pGL3 (firefly luciferase) vector. 24 hours after transfection (time = 0 h), cells were treated with DRB. Total RNA was extracted after various time-points. RNA was reverse-transcribed and RealTime RT-PCR was performed. Renilla luciferase transcript levels were normalized to GAPDH and firefly luciferase mRNAs. Data are expressed as percentage of RNA remaining after DRB addition and are representative of three independent experiments. B) Schematic representation of luciferase constructs carrying C2 fragment and sub-fragments. C) Luciferase activity of the chimeric constructs in SK-N-BE, SH-SY5Y, HEK-293 and MCF-7 cell lines. Cells, transiently co-transfected with the pGL4.71P- constructs (Renilla luciferase) and the pGL3 (Firefly luciferase) vector were harvested 24 hours post-transfection. Luciferase activity of the chimeric constructs normalized as described, is represented as a percentage of the activity observed in cells transfected with pGL4.71P (defined as 100%). Means ± s.d. luciferase values were obtained from at least four independent experiments (* p < 0.01 compared with the corresponding pGL4.71P value). D) mRNA levels of the chimeric transcripts. Total RNA was extracted from the cells harvested 24 hours post-transfection, and the reporter gene mRNA levels were analyzed by RealTime PCR. mRNA levels of the chimeric reporter gene normalized as described, are represented as a percentage of the mRNA levels observed in cells transfected with pGL4.71P (defined as 100%). Means ± s.d. luciferase mRNA values were obtained from at least three independent experiments (* p < 0.01 compared with the corresponding pGL4.71P value).