Stathmin is efficiently knocked out in C57BL/6 and FVB mouse strains.

<p>(A) PCR analyses of genomic DNA extracted from the tail of WT, stathmin heterozygous and KO C57BL/6 and FVB mice. (B) qRT-PCR analyses of stathmin mRNA expression in brain and thymus from 10 weeks old WT and stathmin KO C57BL/6 and FVB mice. (C) qRT-PCR analyses of stathmin mRNA expression in bladder and skeletal muscle from 10 weeks old WT and stathmin KO C57BL/6 mice. (D) and (E) Western Blot analysis of stathmin protein expression in thymus and spleen of 15 weeks old WT and stathmin KO C57BL/6 mice (D) and in mouse embryo fibroblasts (MEF) isolated from 13.5 days old C57BL/6 or FVB embryos (E). Vinculin was used as loading control.</p

Stathmin is not required for tumor onset following DMBA/TPA treatment in mice.

<p>(A) Kaplan Meier curves of tumor-free WT, stathmin heterozygous and KO mice challenged with the 7,12-dimethylbenz[α]antracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) protocol, using as endpoint 20 weeks of treatment. p = n.s., using the Log Rank test. (B) Evaluation of the number of tumors/mice in function of time. <i>p = n.s.,</i> using the Mann Whitney test. (C) Macroscopic and microscopic analyses of papillomas in mice of the indicated genotypes. Typical images are reported. H&E  =  hematoxylin and eosin staining. CK1  =  IHC analysis of cytokeratin 1 expression. Loricrin  =  IHC analysis of loricrin expression.</p

Molecular characterization of papillomas from WT, stathmin heterozygous and KO mice.

<p>(A) DNA sequence analysis, using the Sanger method, of H-Ras in tumors from WT, stathmin heterozygous and KO mice. The T-A substitution in heterozygosis is framed in black. A typical sequence is reported. (B) qRT-PCR analyses of K-Ras4a, K-Ras4b, c-Fos, Egr-1, Jun-B, Cyclin D1 and c-Myc mRNA expression in tumors from mice of the indicated genotype. The horizontal bar within the box indicates the median expression of each gene. (C) Western blot analyses of AKT and MAPK activation in papillomas derived from mice of the indicated genotype. On the right, the box-plot show the ratio between phosphorylated and total ERK (upper panel) and phosphorylated and total AKT (lower panel), in 6 tumors/genotype. <i>p = n.s.,</i> using the Mann Whitney test.</p

Tumorigenic potential of MEF transformed with LgTAg and K-Ras4b^G12V does not depend on stathmin expression.

<p>(A) Western blot analysis showing the expression of papilloma virus Large T Antigen (LgTAg) (upper panel), K-Ras (middle panel) and ERK1/2 (lower panel) in mouse embryo fibroblasts (MEF) of the indicated genotype, transduced with retroviruses encoding for LgTAg and K-Ras4b^G12V. In the first lane (WT), the protein lysate from non-transduced WT MEF was used as negative control for K-Ras and LgTAg expression. (B) Growth curve analysis on two independent K-Ras clones/genotype. Data represent the mean of 3 independent experiments performed in duplicate. (C) Graph reports the analysis of BrdU incorporation in the indicated cell clones, exposed to BrdU for two hours and then fixed and analyzed by immunofluorescence. Data are expressed as percentage of BrdU positive cells respect to the total and represent the mean of 3 independent experiments in which at least 200 cells were counted. (D) Graph reports results from soft agar assay. The numbers of colonies/field (using a 10 × objective) formed by the indicated cell clones in two independent experiments are reported. The black bar indicates the median number of colonies for each cell clone. (E) <i>In vivo</i> growth of WT and stathmin KO MEF transformed with LgTAg + K-Ras4b^G12V, evaluated by measuring tumor volume each week for 5 weeks in 8 mice/genotype. <i>p = n.s.,</i> using the Mann Whitney test. (F) Graph reports the weight of the tumors explanted from mice injected with LgTAg + K-Ras4b^G12V transformed WT and stathmin KO MEF. The black bar indicates the median tumor weight. <i>p = n.s.,</i> using the Mann Whitney test.</p

Histological characterization of 3-methyl-cholanthrene-induced sarcomas.

1<p>Tumors were classified according to their morphology as spindle, pleomorphic or round cell sarcomas. In some cases, multiple morphologic phenotypes were present in the same tumor.</p>2<p>Local Infiltration was rated as follows: 0  =  no tumor infiltration; 1  =  minimal tumor infiltration in the surrounding muscle; 2  =  massive tumor infiltration.</p>3<p>Necrosis was rated as follows: 0  =  no necrosis; 1  =  minimal areas of necrosis; 2  =  large areas of necrosis.</p>4<p>p53 staining was rated as follows: 0  =  no staining; 1  =  faint staining in less than 50% of the cells; 2  =  strong staining in less than 50% of the cells; 3  =  strong staining in more than 50% of the cells. ND  =  not done.</p

SCG10, SCLIP and RB3 are expressed at similar levels in WT and stathmin KO mice.

<p>(A) qRT-PCR analyses of the expression of SCG10, SCLIP and RB3 in brain, thymus and skin papilloma derived from WT and stathmin KO mice. (B) qRT-PCR analyses comparing the mRNA levels of stathmin 1, SCG10, SCLIP and RB3 in brain, thymus and skin papilloma from WT mice. Data represent the mean (±SD) of three mice/genotype (brain and thymus) or four mice/genotype (skin papillomas).</p

Genetic characterization of p27^kip1 and stathmin in controlling cell proliferation in vivo

<div><p>The CDK inhibitor p27^kip1 is a critical regulator of cell cycle progression, but the mechanisms by which p27^kip1 controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27^kip1 binding partner. To get more insights into the in vivo significance of this interaction, we generated p27^kip1 and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27^kip1 null mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27^kip1 null mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27^kip1 to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.</p></div

Additional file 2: Table S2. of N-BLR, a primate-specific non-coding transcript leads to colorectal cancer invasion and migration

Primer sequences used for qRT-PCR, RACE, siRNAs, and vector construction in this study. (XLSX 11 kb