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. 2016 Feb 29;44(6):2475–2490. doi: 10.1093/nar/gkw118

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© The Author(s) 2016. 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-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 8. — Lovastatin increases the poly(A) tail length of p27^kip1 mRNA and its protein output via QKI and PAPD4. HeLa cells were transfected with siRNA against either Luciferase, QKI or PAPD4. Forty-eight hours later, HeLa cells were harvested at the specified times after the addition of lovastatin. (A) The total cell lysate was analyzed by western blotting with the indicated antibodies. (B) The amount of p27^kip1 protein was measured and normalized to the GAPDH protein. The protein level at the 0 h time point was defined as 100%. The results were derived from three independent experiments and shown as the means ± SD. *P < 0.05. (C) The poly(A) tail length of the endogenous p27^kip1 mRNA was analyzed using an RL-PAT assay and the amplified cDNA was detected by Southern blotting. To quantitatively estimate distribution of the poly(A) tail length, the mRNAs were digested with RNase H in the presence of oligo(dT) to localize deadenylated (A0) mRNA and the corresponding region of the mRNA signal was designated as poly(A)⁻; the upper portion was designated as poly(A)⁺ as indicated. (D) The amount of poly(A)⁺ portion was measured and normalized to the poly(A)⁻ portion. The poly(A) tail level at 0 h time point was defined as 100%. The results were derived from three independent experiments and shown as the means ± SD. **P < 0.01. (E) The amount of p27^kip1 mRNA was measured by RT-PCR and was normalized to that of GAPDH mRNA. The mRNA level at the 0 h time point was defined as 100%. The results were derived from three independent experiments and shown as the means ± SD.