(A) Three independent preparations of total RNA from early-passage (Young, pdl 25) and from late-passage (Senescent, pdl 54) WI-38 cells were subjected to microarray analysis (Exiqon, Materials and Methods). Thirty-one miRNAs showed reduced levels in the senescent cultures (by two-fold in at least two samples), 9 miRNAs showed increased levels (Supplemental Fig. S2). (B) The differential expression of ten downregulated miRNAs was validated using RT-qPCR analysis; the relative levels of each miRNA in Y and S WI-38 cells are shown (means+SEM from three independent experiments).

Late-passage WI-38 cells (pdl 51) were transfected with pre-miR-24 or Ctrl. siRNA (100 nM) and 48 hr later (A) RT-qPCR was used to measure the levels of p16 mRNA (normalized to GAPDH mRNA levels, left) and the levels of miR-24 (normalized to 18S rRNA levels, right), and (B) the levels of p16 and loading control α-Tubulin were monitored by Western blotting, and quantified (%) by densitometry. Early-passage (pdl 20) WI-38 cells were transfected with AS-miR-24 or Ctrl. siRNA (100 nM) and 48 hr later (C) RT-qPCR was used to measure the levels of p16 mRNA (normalized to GAPDH mRNA levels, left) and the levels of miR-24 (normalized to 18S rRNA levels, right), and (D) p16 and loading control α-Tubulin levels were assessed by Western blotting, and quantified by densitometry. (E) p16 mRNA levels in sucrose gradients were calculated as described for Fig. 1C. (A) and (C) show the means+SD from 3 independent experiments.

(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.

Schematic (left) and names (center) of the EGFP and chimeric EGFP-p16 reporter plasmids tested (Materials and Methods). By 48 hr after co-transfection of HeLa Tet-off cells with Ctrl. siRNA or AS-miR-24, along with the plasmids indicated, the levels of EGFP expressed from each reporter vector was assessed; shown are representative Western blotting signals and quantification (Fold, AS-miR-24 vs. Ctrl. siRNA) of EGFP reporter levels in each transfection group after normalization to α-tubulin signals.

(A) The levels of mRNAs encoding cyclin D1, p16, and control housekeeping GAPDH were measured by RT-qPCR, normalized to 18S rRNA levels and plotted as fold levels relative to the levels in pdl 20 HDFs. (B) Western blot analysis of p16 and loading control β-Actin levels in young (Y, pdl 20) and senescent (S, pdl 54) WI-38 HDFs. (C) The polysomal association of mRNAs encoding p16 (left) or GAPDH (right) in Y and S WI-38 cells was tested by fractionating cytoplasmic lysates through sucrose gradients and measuring mRNA abundance by RT-qPCR in each of the 10 resulting fractions (insets). The relative abundance of p16 and GAPDH mRNAs in each gradient fraction is shown as a percent of the total mRNA. NB/NT (‘not bound/not translated’) fractions with mRNA devoid of translational components; LMW, HMW, low and high molecular weight polysome fractions, respectively.

(A) Top, p16 mRNA depicting the two locations (in CR and 3′UTR) where miR-24 is predicted to bind. Bottom left, miR-24 levels were measured in WI-38 cells by RT-qPCR using miR-24-specific primers, and normalized to 5S rRNA levels. Bottom right, PCR-amplified products (∼100-bp), visualized on 1.5% agarose gels, were absent from RT- reactions; MWM, DNA molecular weight markers. (B) Young (pdl 20) or Senescent (pdl 54) WI-38 cells were left utnreated (Untr.) or treated with puromycin (Puro., 200 µM, 1 hr) and cytoplasmic lysates were prepared and fractionated through sucrose gradients. The presence of miR-24 was measured in each fraction, calculated as a percentage of the total miR-24, and plotted using a semilogarithmic scale. The data are representative of 3 independent experiments. (C) The relative abundance (fold) of miR-24 in each fraction was compared between Y and S cells. In fractions 1–5 (black bars), miR-24 was comparable or more abundant in S than in Y cells; in fractions 6–10 (gray bars), miR-24 was more abundant in Y than in S cells. All bars represent the means+SD from 3 independent experiments.

(A) Cells were left untreated (Untr.) or treated with puromycin (Puro., 200 µM, 1 hr); cytoplasmic lysates were prepared and fractionated through sucrose gradients. Top, representative miR-24 levels in each fraction, visualized after RT-PCR (30 cycles) and electrophoresis (1.5% agarose). Bottom, miR-24 levels in each fraction, quantified by RT-qPCR analysis and represented as % of the total miR-24 (semilogarithmic scale). (B) Representative gradient profiles. (C) By 48 hr after transfection of pre-miR-24 or control (Ctrl.) siRNA (100 nM), miR-24 and U6 snRNA levels were visualized on agarose gels (left), quantified by RT-qPCR, and normalized to 5S (right). Data (means+SD) represent the fold differences in miR-24 levels between the transfection groups. (D) HeLa cells were transfected with a plasmid expressing either pTRESNeo (V) or pIRESNeo-HA-Ago1 (HA-Ago1); the specific IP of HA-Ago1 was assessed by Western blot analysis of the IP reaction products. Forty-eight hr after transfection of HeLa cells with plasmid (V or HA-Ago1) and either control (siRNA) or pre-miR-24 RNA, RT-qPCR analysis was used to test the association of p16 mRNA with the RISC complex in the V and HA-Ago1 groups after HA IP. Graphs show the means+SD from 3 independent experiments.

(A) Cells were transfected with either a control (Ctrl.) siRNA or antisense (AS) miR-24 (100 nM) and the levels of mRNAs encoding p16, and controls GAPDH (for normalization) and SDHA were calculated by RT-qPCR and represented as the means+SD from 3 independent experiments; the levels of miR-24 and U6 RNA were also measured. (B) Representative Western blot analysis of p16 (and loading control α-Tubulin) levels in each transfection group. p16 signals were measured by densitometry and represented as % p16 levels relative to the control transfection group. (C) The levels of miR-24 were measured by RT-qPCR in each of the fractions (Materials and Methods) and shown as relative miR-24 levels in the AS-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). Note that the abundance of miR-24 was preferentially reduced in fractions 1 and 2 (left), but the remaining miR-24 was found distributed among all of the fractions of the polysome gradient (right). Data are representative from three independent experiments yielding similar results. (D) The levels of p16 and control GAPDH mRNAs on polysome gradients were measured and represented as explained in the legend of Fig. 1C. (E) Nascent translation of p16 or (control) GAPDH was monitored following incubation of cells with L-[³⁵S]methionine and L-[³⁵S]cysteine for 15 min; after immunoprecipitation (IP) using either anti-p16 (left) or anti-GAPDH (right) antibodies along with control IgG, the incorporation of radiolabeled amino acids into newly synthesized p16 and GAPDH polypeptides was visualized after SDS-PAGE and quantified using a PhosphorImager; the % change in p16 nascent translation is shown.