Figure 4

U0126 does not alter bim mRNA stability in sympathetic neurons. (A) Sympathetic neurons were treated with actinomycin-D at 0.1 μg/ml in the presence of NGF. The cells were either maintained in medium containing NGF (-U0126) or treated with U0126 at 10 μM in the presence of NGF (+U0126). Actinomycin-D and U0126 were added to the cells together and then total RNA was isolated at time point 0 hours, 2 hours, 4 hours, 8 hours and 16 hours. The level of bim mRNA was analysed by q-PCR relative to the level of the mRNAs encoded by the house-keeping genes Hprt1 and Gapdh. Bim mRNA levels relative to Hprt1 are shown here. The data is presented as the mean ± S.E., n = 5. There is no significant difference in the stability of bim mRNA in sympathetic neurons treated with or without U0126 over 16 hours. (B) Sympathetic neurons were treated as in (A) but with the following modification: U0126 was added to the cells for 16 hours prior to the addition of actinomycin-D and U0126 together (for +U0126 conditions). Following addition of actinomycin-D, total RNA was isolated at time point 0 hours, 2 hours, 4 hours, 8 hours and 16 hours. The level of bim mRNA was analysed by q-PCR relative to the house-keeping genes Hprt1 and Gapdh. Bim mRNA levels relative to Hprt1 are shown here. The data is presented as the mean ± S.E., n = 3. There is no significant difference in the stability of bim mRNA in sympathetic neurons treated with or without U0126 over 16 hours. (C) Q-PCR data from (B) with raw bim mRNA levels at timepoint 0 (+U0126 and -U0126 bim mRNA levels have not been normalised to 1). Treatment with U0126 for 16 hours elevates bim mRNA levels significantly (p = 0.021), but does not significantly alter the half life of the bim mRNA. (D) Sympathetic neurons were either maintained in medium containing NGF (-actinomycin-D) or treated with actinomycin-D (+actinomycin-D) at 0.1 μg/ml in the presence of NGF. After 16 hours, the cells were treated with U0126 (together with actinomycin-D for +actinomycin-D conditions) and then total RNA was isolated at time point 0 hours, 2 hours, 4 hours, 8 hours and 16 hours. The level of bim mRNA was analysed by q-PCR relative to Hprt1 and Gapdh. Bim mRNA levels relative to Hprt1 are shown here. The data is presented as the mean ± S.E., n = 3. Treatment with U0126 for 16 hours, in the absence of actinomycin-D, elevates bim mRNA levels significantly (p = 0.036).