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

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

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

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

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

Additional file 3: of KAT3B-p300 and H3AcK18/H3AcK14 levels are prognostic markers for kidney ccRCC tumor aggressiveness and target of KAT inhibitor CPTH2

Immunostaining of K1 papyllary thyroid cells with p300 antibody after 72 h of treatment with CPTH2 (100 μM) compared to untreated and DMSO controls. Apoptotic percentage of 786-O cells treated with proteasome inhibitor MG-132 (1 h) and then incubated in DMSO w/w CPTH2 shows no significative changes of the apoptotic profiles compared to the untreated controls. p300 immunostaining of 786-O cells were pretreated for 1 h with proteasome inhibitor MG132, then grow in DMSO w/w CPTH2 for 18 h suggest that there is no significative proteolysis of p300 upon inhibition of the proteasome. (TIFF 30444 kb

Additional file 2: of KAT3B-p300 and H3AcK18/H3AcK14 levels are prognostic markers for kidney ccRCC tumor aggressiveness and target of KAT inhibitor CPTH2

786-O cells grown in DMSO w/w CPTH2 for 24 and 48 h and immunostained with anti-Ki67 and anti-cyclin D1 show that CPTH2 treatment does not affect cell cycle progression. (TIFF 24905 kb

Venn diagram and associated table describing mRNA expression profile patterns in the three classes.

<p>Venn diagram (<b>A</b>) representing differentially expressed mRNAs observed in the comparison among the three classes (normal, primary tumor, metastatic lesion) for patients of 50 yrs and younger, up or down facing arrow indicate the expression level change. Tables show: the 7 mRNAs identified by the comparison between Normal <i>vs</i> Tumor (<b>B</b>); 1 mRNA differentially expressed only in Tumor vs Mets (<b>C</b>); 4 mRNAs differentially expressed in Normal vs Mets (<b>D</b>); 2 mRNAs commonly deregulated in the comparisons Tumor vs Mets and Normal vs Mets (<b>E</b>).</p

Overall Survival miRNA signature and Distant Disease-Free Survival (DDFS) signature.

<p>Overall survival (OS) of TNBC patients of 50 yrs and younger patients due to differentially expressed miRNAs in the three classes. (<b>A</b>) Heat map representing miRNA profiles of 75 tumor samples using average linkage clustering and Spearman Rank method as distance metrics. Bar above the dendrogram identifies 39 high risk samples shown in orange and 36 low risk cases in yellow. Samples are shown in columns, miRNAs in rows. Heat map represents relative miRNA expression as indicated in the blue to red key bar at the top. (<b>B</b>) Hazard ratios of protective and risky miRNAs. (<b>C</b>) Overall Survival miRNAs signature. Distant Disease-Free Survival (DDFS) miRNA signature of 50 yrs and younger. (<b>D</b>) Heat map representing miRNA profiles of 75 tumor samples using average linkage clustering and Spearman Rank method as distance metrics. Bar above the dendrogram identifies 37 high risk samples shown in orange and 38 low risk cases in yellow. Samples are shown in columns, miRNAs in rows. Heat map represent relative miRNA expression as indicated in the key bar at the top. (<b>E</b>) miRNAs predicting protection from or susceptibility to early recurrence. (<b>F</b>) Kaplan-Meier estimates of DDFS according to the seven-miRNA signature.</p