Western blot analysis was carried out as described previously Huang and Pardee, The blot was probed with rabbit polyclonal antibody to p21 Cat. The blot was subsequently stripped and reprobed with rabbit polyclonal antibody to actin Cat. A; Sigma , which served to normalize the amount of loaded protein. The washed blot was exposed to Kodak X-AR film for detection of annealed probe. The gel was then dried and exposed to an X-ray film. Sp1-luc contains the promoter driven by three consensus Sp1 binding sites from the SV40 promoter, whereas mtSp1-luc contains three mutant Sp1 sites.
Protein concentrations in cell lysates were measured using the BioRad protein assay reagent, to normalize the luciferase activities among samples. Transfections were done in duplicate, and repeated three times. AACR 40 : , abstr. USA 95 : — Huang L and Pardee AB. Moustakas A and Kardassis D. USA 93 : — Struhl K. Cell Biol. Wolffe AP. Download references. You can also search for this author in PubMed Google Scholar. Reprints and Permissions. Huang, L. Oncogene 19, — Download citation. Received : 07 July Revised : 21 September Accepted : 25 September Published : 23 November Issue Date : 23 November Anyone you share the following link with will be able to read this content:.
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Download PDF. Abstract Suberoylanilide hydroxamic acid SAHA is a novel histone deacetylase inhibitor with high potency in inducing differentiation of cultured murine erythroleukemia cells. Introduction Suberoylanilide hydroxamic acid SAHA is a prototype of hydroxamic acid-based second generation hybrid polar compounds which are developed as cytodifferentiating agents for potential cancer therapy Richon et al. Sikder, H. Id proteins in cell growth and tumorigenesis.
Cancer Cell 3 , — Burrell, R. The causes and consequences of genetic heterogeneity in cancer evolution. Cha, H. Georgakopoulou, E. Specific lipofuscin staining as a novel biomarker to detect replicative and stress-induced senescence. A method applicable in cryo-preserved and archival tissues. Aging 5 , 37—50 Evangelou, K. Papachristou, E. Proteome Res. Ramirez-Gonzalez, R. StatsDB: platform-agnostic storage and understanding of next generation sequencing run metrics.
Langmead, B. Fast gapped-read alignment with Bowtie 2. Methods 9 , — Li, H. Bioinformatics 25 , — Chen, K. BreakDancer: an algorithm for high-resolution mapping of genomic structural variation. Methods 6 , — Venkatraman, E.
A faster circular binary segmentation algorithm for the analysis of array CGH data. Bioinformatics 23 , — Download references. We would like to thank A. Kotsinas, K. Evangelou, T. Liloglou and A. Georgakilas for their valuable support to this work. Dutta for providing the vectors with the wt and PIP mutated domain of p21, G.
Lygerou for the secondary antibodies employed in the IF analyses. We thank R. Allsopp, D. Emma J. Julian Blow. Akshay K. You can also search for this author in PubMed Google Scholar.
Correspondence to Jiri Bartek or Vassilis G. PBGD: Porphobilinogen deaminase house-keeping gene ii. Representative immunoblots that validate the proteome. Actin serves as a loading control.
Activation of the senescence barrier occurs at approximately day 3 of induction in both cellular systems and increases gradually, reaching its highest value at around day 10, while no signs of senescence are evident in untreated cells grown for the same time period as corresponding graphs depict. Diagram describing cell fractionation experimental algorithm. Comet assays showed DNA breaks in cells infected with the indicated constructs see also Fig. Red lines in magnifications of insets label comet moment tails for length comparison.
FACS analysis of the corresponding treatments. Source data can be found in Supplementary Table Saos2- and iii. Timeline of the experimental procedure is also depicted i.
After the indicated treatments, cells were fixed and stained with antibodies against BrdU without DNA denaturation to selectively detect nascent-strand ssDNA. Absence of p73 promoter methylation and genetic loss at TP73 locus 1p S5, S6; Supplementary Table 4. Bioinformatic analysis employing Ingenuity software revealed potential factors that regulate p73 expression and activity.
In turn, p73 can also transcriptionally induce EGR-1exprresion, forming a positive feed-back loop. Decreased levels of EGR-1 possibly represent the main reason for low p73 expression Additionally, high levels of PRKACB decreases p73 transactivation and intramolecular interaction abilities 2 , counteracting the ability of high HECW2 expression to stabilize p73 via mono-ubiquitination 1. Expression status of genes associated with cancer progression see also Supplemental Table 8. Uncropped images of blots are shown in Supplementary Fig.
In total 41 aberrations were found involving all chromosomes except 9, 12, 14 and The aberrations included 19 gains and 22 losses Supplemental Table 5. The majority of aberrations were concentrated in chromosomes 3, 10 and X Supplemental Table 5. CIN:chromosomal instability. Telomeric regions were found to be most frequently affected by fusions, translocations and tandem duplications of large chromosome segments.
As unidentified ones were categorized the non-telomeric, non centromeric genomic rearrangements in which the cytogenetic bands of their breakpoints remained obscure. Arrows and dashed rectangles indicate representative non-clonal random structural rearrangements unique anomalies encountered in a single cell.
Cells 3 and 5, belong to a second subclone of the control cells that is characterized by a deletion of a rearranged chromosome In addition, cells 2, 4 and 9, have lost a marker translocation der 9 t 5;9 that was replaced by a deletion 9p and acquired clonally an extra translocated der 22 t 20; Cells 5 and 7 represent two different endoreduplicated ON subclones, characterized by unique structural abnormalities of chromosomes 7, 15 and 6 respectively.
Data from all replicates for each application were averaged before comparison. Novel chromosomal rearrangements and microhomology regions related to breakpoints in a Saos2 and b Li—Fraumeni cells.
Data from two biological replicates are depicted. Asterisk a denotes breakpoint that does not encompass a micro-homology. Continuous red line denotes position of breakpoints. A: Relative expression of all measured genes at each depicted time-point as compared to non-induced cells OFF. Dashed lines depict ineffective pathway.
Reprints and Permissions. Galanos, P. Chronic pindependent p21 expression causes genomic instability by deregulating replication licensing. Nat Cell Biol 18, — Download citation. Received : 28 October Accepted : 19 May Published : 20 June Issue Date : July Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. The gene is up-regulated in mouse fibroblasts in response to serum restimulation but the kinetics and levels of induction differ between wild-type and mutant cells.
Expression of p21 message following serum restimulation is superinducible by cycloheximide in wild-type but not in pdeficient cells. The increases in p21 mRNA are reflected in changes in p21 protein levels. Regulation of the mouse p21 promoter by p53 depends on two critical pbinding sites located 1.
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