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. 2014 Oct 28;42(20):12555–12569. doi: 10.1093/nar/gku1033

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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Resveratrol reduces the stability of IL-8, iNOS and TNF-α mRNA in human DLD-1 cells in a KSRP-dependent manner. DLD-1 cells were stably transfected with constructs resulting in the intracellular expression of KSRP siRNA (siKSRP) or Luc siRNA (siLuc, control). (A) Expression of KSRP (KSRP) in siKSRP and siLuc cells was analyzed by western blot experiments using a specific anti-KSRP antibody. For normalization, the expression of β-tubulin (β-tubulin) was analyzed in parallel using a specific anti-β-tubulin antibody. Shown is one of four representative western blots. (B–D) DLD1 siLuc or siKSRP cells were treated with CM for 2 h (B and D) or 4 h (C). Note that 30 μM resveratrol was added and 30 min later the cells were treated with DRB (25 μg/ml). After 10, 20, 30, 45 min (B and D) or 2, 4 and 6 h (C) RNA was isolated and the expression of IL-8, iNOS, TNF-α and GAPDH (for normalization) mRNA was measured by qRT-PCR. The IL-8, iNOS or TNF-α mRNA expression was normalized to the GAPDH mRNA expression. (B) The relative IL-8 mRNA expression after 2 h CM was set to 100%. Shown are the mean ± SEM of n = 8–10 analyses (###P < 0.001; ##P < 0.01; #P < 0.05; ns_#, not significant versus siKSRP cells not treated with resveratrol, two-way ANOVA). The half-lifes of the IL-8 mRNAs under the different conditions are: siLuc CM 10.09 ± 0.70 min; siKSRP: CM 19.76 ± 4.18 min, siKSRP CM + Res 20.88 ± 3.29 min. (C) The relative iNOS mRNA expression after 4 h CM was set to 100%. Shown are the mean ± SEM of n = 10–12 analyses (###P < 0.001; #P < 0.05; ns_#, not significant versus siKSRP cells not treated with resveratrol; two-way ANOVA). The half-lifes of the iNOS mRNAs under the different conditions are: siLuc CM 4.31 ± 0.75 h; siKSRP: CM 7.60 ± 2.88 h, siKSRP CM + Res 7.83 ± 2.36 h. (D) The relative TNF-α mRNA expression after 2 h CM was set to 100%. Shown are the mean ± SEM of n = 12–14 analyses (#P < 0.05; ns_#, not significant versus siKSRP cells not treated with resveratrol; two-way ANOVA). The half-lifes of the TNF-α mRNAs under the different conditions are: siLuc CM 14.85 ± 0.94 min; siKSRP: CM 20.17 ± 4.29 min, siKSRP CM + Res 24.81 ± 4.37 min.