FIGURE 2.

HuR plays a critical role in CsA-induced VEGF mRNA stability. A, 786-0 cells were transfected with either HuR siRNA (25 nmol/liter) or control siRNA for 72 h. The cells were then preincubated with actinomycin D (5.0 μg/ml) and treated with CsA (5.0 μg/ml) or the vehicle alone for different time intervals (0–6 h). The cells were harvested at designated time points, and total RNA was isolated and reverse transcribed. The fold change in VEGF mRNA expression was measured by real-time PCR as described under “Experimental Procedures” and is represented as percentage decrease in mRNA levels using a semilogarithmic scale. The mRNA level at zero time point (before CsA treatment) was considered as 100%. Results are representative of three independent experiments from triplicate readings of the samples. Points, average value of VEGF mRNA expression; error bars, S.D. ANOVA indicated a significant change in percent VEGF mRNA expression during the 6-h period with highly significant slope differences between the treatment groups (F = 197.1, p < 0.0001). Control siRNA + CsA group showed a slower rate of decline compared with control siRNA group (t = 2.51, p = 0.019). HuR siRNA alone showed a faster rate of decline in mRNA compared with control siRNA group (t = 5.11, p < 0.0001). Slopes for mRNA expression were significantly more rapid in HuR siRNA (t = 7.61, p < 0.0001) and HuR siRNA + CsA group (t = 8.88, p < 0.0001) compared with control siRNA + CsA group. B, knockdown of HuR (36 kDa) in 786-0 cells was confirmed by Western blot analysis after 72 h of siRNA transfection. The lanes were grouped from different parts of the same gel and same blot. Results are representative of two different experiments.