Increasing miR-24 reduces p16 levels in HeLa cells.
(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.