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Increasing miR-24 reduces p16 levels in HeLa cells.

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posted on 2008-03-26, 01:21 authored by Ashish Lal, Hyeon Ho Kim, Kotb Abdelmohsen, Yuki Kuwano, Rudolf Pullmann Jr., Subramanya Srikantan, Ramesh Subrahmanyam, Jennifer L. Martindale, Xiaoling Yang, Fariyal Ahmed, Francisco Navarro, Derek Dykxhoorn, Judy Lieberman, Myriam Gorospe

(A) HeLa cells were transfected with either Ctrl. siRNA or pre-miR-24 (100 nM) and 48 hr later cytoplasmic lysates were prepared and fractionated through sucrose gradients. The levels of miR-24 were then measured by RT-qPCR in each of the fractions (Materials and Methods) and shown as relative (fold) miR-24 levels in the pre-miR-24 transfection group relative to the levels in the Ctrl. siRNA transfection group (left) and also as a percentage of the total miR-24 in each of the two transfection groups (right). (B) HeLa cells were transfected and processed as described in panel (A) except that the comparisons were made between populations transfected with AS-miR-24 and Ctrl. siRNA. The levels of mRNAs encoding p16 and housekeeping controls GAPDH (for normalization) and SDHA were calculated by RT-qPCR and represented (means+SD from 3 independent experiments) as fold differences relative to control transfected cells. (C) Representative Western blot analysis of p16 (and loading control α-Tubulin) levels in each transfection group. p16 protein signals were measured by densitometry and represented as a percent of the p16 levels in the control transfection group. (D) Representative distribution of p16 and GAPDH mRNAs on polysome gradients, calculated and shown as explained for Fig. 1C.


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