Knock-down of CDCA2, ID4 or both does not induce premature senescence in PDL 33 IMR90 cells.

<p>A) siRNAs designed to target the coding sequence of CDCA2 or ID4 were transfected, individually or as a mix, in PDL 33 IMR90 cells to knock-down the expression levels of target genes. After 72 h, transfected cells were incubated with BrdU for 4 h, then coverslips were fixed, incubated with a primary anti-BrdU antibody, washed and incubated with a secondary fluorescein-conjugated antibody, counterstained with Hoechst-33258 and counted by immunofluorescence. Counts of at least 1,000 cells were averaged and expressed as fold changes±SD, with respect to scrambled transfected cells (*** p<0.001). B) siRNA transfected cells were subcultivated for 10 days and stained for SA-β-gal. Counts of at least 300 cells were averaged and expressed as fold changes±SD, with respect to scrambled transfected cells.</p

Strategy to identify putative targets of SAmiRs.

<p>A) The 3’UTR sequences of the 139 mRNAs down-regulated in replicative senescent fibroblasts and reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098669#pone.0098669.s005" target="_blank">Table S1</a> were analyzed with four different target prediction algorithms. This <i>in silico</i> analysis revealed 30 putative SAmiR targets: 20 of SAmiR-494, 7 of SAmiR-486-5p and 3 common to both SAmiRs. B) List of the 30 predicted target genes of both SAmiR-494 or SAmiR-486-5p. In grey, the three putative targets common to both SAmiRs.</p

Expression profile of putative targets upon the induction of replicative or stress-induced senescence.

<p>The expression levels of the 26 candidates were measured by Real Time PCR in replicative (<b>RS</b>: IMR90 cells at PDL 58), etoposide- (<b>EIS</b>: PDL 33 IMR90 cells treated with 20 µM etoposide for 24 h and then subcultivated for additional 10 days) and DEM-induced (<b>DIS</b>: PDL 33 IMR90 cells treated with 150 µM DEM on alternate days for 10 days) senescent cells. The mRNA relative expression was calculated by assigning the arbitrary value 1 to the amount found in young or DMSO-treated cells. SD is used to refer to the values obtained in 2 different experiments. Results showed that 7 mRNAs, highlighted by a star, resulted significantly (p<0.05) down-regulated, with a cut-off≥2 folds, in all conditions.</p

Adoptive expression of CDCA2 promotes cell cycle progression in Etoposide-Induced Senescence.

<p>A) CDCA2 and ID4 coding sequences were transfected in PDL 33 IMR90 cells. Cells transfected with CMV-NEO plasmid were used as control. After 24 h, transfected cells were treated with 20 µM etoposide or 150 µM DEM for 24 h and then incubated with BrdU for 4 h. Coverslips were then fixed, incubated with primary anti-BrdU and secondary fluorescein-conjugated antibodies, counterstained with Hoechst-33258 and counted by immunofluorescence. Counts of at least 800 cells were averaged and expressed as fold changes±SD, with respect to control transfected cells (***p<0.01). B) and C) PDL 33 IMR90 cells were transfected with the coding sequence of CDCA2. After 24 h, transfected cells were treated with 20 µM etoposide for 24 h and then were collected to obtain protein extracts. Cell extracts from CMV-neo over-expressing cells served as control. Western Blot analysis was used to detect the levels of phosphorylated ATM (p-ATM Ser1981), ATM, phosphorylated p53 (p-p53 Ser15), p53, p21^Cip1 and CDCA2 in the cell lysates. β-actin was used as a loading control.</p

Expression profile of putative targets upon SAmiRs ectopic expression and validation of CDCA2 and ID4.

<p>A) Expression levels of SAmiR-494 and SAmiR-486-5p putative targets 2 days after the transfection of the cognate SAmiR pre-miR in PDL 33 IMR90 cells. mRNA levels were measured by Real Time PCR and mRNA relative expression was calculated by assigning the arbitrary value 1 to the amount found in control cells transfected with a scramble pre-miR. SD refers to the values obtained in 3 different experiments and the difference was significant (** p<0.01; * p<0.05). The quantification of the expression levels of SAmiRs after their ectopic over-expression is reported in Panel A of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098669#pone.0098669.s002" target="_blank">Figure S2</a>. B) Luciferase constructs bearing the normal 3’UTRs (N), reverse 3’UTRs (R) or mutated 3’UTRs (M) of CDCA2 and ID4 were transfected in HEK293 cells together with the cognate pre-miR or control pre-miR. The normal and reverse 3’UTR of OLFM4 were used as positive control. Luciferase levels were reported as fold changes compared to the values measured in control pre-miR transfected cells, after normalization with Renilla luciferase activity. SD refers to the values obtained in 3 different experiments (* p<0.01). C) Wild type seed regions of SAmiR-494 and SAmiR-486-5p, respectively present into the 3’UTRs of CDCA2 and ID4, compared to the mutated seed regions used for luciferase assays. D) Western blot analysis of CDCA2 in IMR90 cells over-expressing SAmiR-494 (pre-miR) or a specific SAmiR-494 inhibitor (anti-miR). E) Western blot analysis of ID4 in IMR90 cells over-expressing SAmiR-486-5p (pre-miR) or a specific SAmiR-486-5p inhibitor (anti-miR). In both cases, the proteins resulted suppressed by the SAmiR over-expression, as well as they resulted up-regulated by the anti-miR transfection, if compared to the control scramble transfected cells. The quantification of the expression levels of SAmiRs after their ectopic over-expression in D and E is reported in Panel B of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098669#pone.0098669.s002" target="_blank">Figure S2</a>.</p