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

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 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

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 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

Disclosing the Interaction of Carbonic Anhydrase IX with Cullin-Associated NEDD8-Dissociated Protein 1 by Molecular Modeling and Integrated Binding Measurements

Human Carbonic Anhydrase (hCA) IX is a membrane-associated member of the CA enzyme family, involved in solid tumor acidification. This enzyme is a marker of tumor hypoxia and a prognostic factor for several human cancers. In a recent paper, we showed that CA IX interacts with cullin-associated NEDD8-dissociated protein 1 (CAND1), a nuclear protein involved in gene transcription and assembly of SCF ubiquitin ligase complexes. A functional role for this interaction was also identified, since lower CA IX levels were observed in cells with decreased CAND1 expression <i>via</i> shRNA-mediated interference. In this paper, we describe the identification of the structural determinants responsible for the CA IX/CAND1 interaction by means of a multidisciplinary approach, consisting of binding assay measurements, molecular docking, and site-directed mutagenesis. These data open a novel scenario in the design of anticancer drugs targeting CA IX. Indeed, the knowledge of the structural determinants responsible for the CAND1/CA IX interaction provides the molecular basis to design molecules able to destabilize it. Due to the proposed function of CAND1 in stabilizing CA IX, these molecules could represent an efficient tool to lower the amount of CA IX in hypoxic cancer cells, thus limiting its action in survival and the metastatic spread of tumors