ABSTRACT: To evaluate the effect on gene expression by shRNA-58335, we evaluated gene expression by microarray analysis. Gene expression was measured in Huh-7 cells stably expressing shRNA-58335 or control shRNA after infection with adenovirus expressing p53 (Ad-p53) or LacZ (Ad-LacZ).
Project description:To evaluate the effect on gene expression by lincRNAs, we knocked down three lincRNAs and evaluated the gene expression by microarray analysis. Gene expression was measured in Hep3B cells transfected with siRNAs targeting the indicated lincRNAs or negative control siRNA and infected with adenovirus expressing p53 (Ad-p53) or LacZ (Ad-LacZ).
Project description:To characterize the molecular features of p53 signaling pathway, we generated the gene-expression profiles of p53-introduced U373MG cells by genome-wide cDNA microarrays Keywords: time course U373MG cells were infected with 20 MOI of Ad-p53 or Ad-LacZ. mRNA from infected cells were collected at 0h, 6h, 12h, 24h and 48h. we used the RNA mixtures from Ad-LacZ infected cells as a reference of cDNA microarray analysis.
Project description:To evaluate the effect on lincRNA expression by p53 family members, we overexpressed p53 family members in H1299 cells and evaluated the lincRNA expression by microarray analysis. lincRNA expression was measured in H1299 cells infected with adenovirus expressing LacZ, p53, p63a, p63g, p73a and p73b.
Project description:Although mutations in p53 are infrequent in human melanoma, its function is abnormal. In this study, whole genome bead arrays were used to examine the expression of p53 target genes in extracts from 6 melanoma cell lines, compared to extracts derived from diploid human melanocytes and fibroblasts, to provide a global assessment of aberrant p53 function. The expression of these genes was also examined in extracts derived from melanocytes and melanoma cell lines in which p53 expression had been inhibited using shRNA and compared to cells that had been transduced with a control shRNA. Total RNA extracted from 18 samples was analysed representing duplicates of 6 melanoma cell lines, 1 melanocyte cell line and 2 fibroblast cell lines. Melanoma cell lines were compared to normal cell lines. In addition, IgR3, Mel-RM and melanocyte cell lines were transduced with either control shRNA or p53 shRNA to evaluate the effect of p53 on its target genes. Cell lines transduced with control shRNA were compared to cell lines transduced with p53shRNA. Duplicates were analysed.
Project description:Time point (H0, H24, H72) expression data from an EBV cell line obtained from a Fanconi Anemia patient (FANCA) transduced with a control shRNA (sh Ctrl) (n=3) or a shRNA targeting p53 (sh p53) (n=3) and treated with a brief MMC pulse.
Project description:NFBD1 (nuclear factor with BRCT domains 1), also known as MDC1 (mediator of DNA damage signaling 1), is a protein involved in the ATM signaling pathway in response to DNA damage. In addition to a role in signaling, NFBD1 possesses transactivation activity, suggesting that it may influence transcription. Furthermore, NFBD1 affects p53-mediated transcription in the presence of the DNA damaging agent adriamycin. Our goal was to determine if NFBD1 affects global gene expression with or without ionizing radiation-induced DNA damage. Moreover, we sought to separate p53-dependent from p53-independent events. Illumina microarray analysis was carried out on RNA extracted from cells treated with and without NFBD1 shRNA and/or p53 shRNA in the presence and absence of DNA damage. Surprisingly, we found that the expression of NFBD1-regulated genes was changed in both the presence and absence of DNA damage. Furthermore, most NFBD1-regulated genes were regulated independently of p53. The identification of NFBD1-regulated genes was confirmed with a second NFBD1-directed shRNA sequence. The role of NFBD1 in global gene expression both in the presence and absence of ionizing radiation was determined. p53-dependent and p53-dependent events regulated by NFBD1 depletion were studied. U2OS cells were treated with control, NFBD1 shRNA, and/or p53 shRNA-expressing retrovirus. Cells were then treated with ionizing radiation and total RNA was isolated for Illumina microarray analysis.
Project description:Micorarray analysis was performed on RNA samples from hippocampal cultures infected with either Ad-aCaN or Ad-LacZ or uninfected. Each sample was applied to its own GeneChip (Rat RG-U34A; n=7-9 chips/group). Chips were then processed and scanned using Agilent Affymetrix GeneArray Scanner. MAS5 was used to determine signal intensity and presence/absence calls for the data.
Project description:The human TP53 gene is frequently mutated in tumors and cell lines. Unlike other tumor suppressors that are commonly inactivated by deletions or nonsense mutations, the majority of p53-mutations are missense point mutations that result in the expression of a full-length protein with an altered amino acid that has lost sequence specific DNA-binding. Expression of mutant p53 (mutp53) confers advantages to tumor cells and transcriptional regulation of several genes mediating the beneficial effects has been shown to play a role. However, molecular mechanisms of transcriptional regulation by mutp53 are still poorly understood. We used the glioblastoma-derived U-251 MG human cell line endogenously expressing mutp53 protein (R273H mutation) to analyze gene expression profiles on Agilent Whole Human Genome Microarray after transient and stable depletion of mutp53 expression. Gene expression data was correlated with a ChIP study on a custom tiling array to understand the contribution of endogenously expressed mutp53 to transcriptional regulation. This series of microarray experiments contains the gene expression profiles of glioblastoma-derived U-251 MG human cell lines engineered to constitutively express a p53-specific shRNA or scrambled control shRNA. To reverse the effect of mutp53 depletion, stable clones were modified by stable integration of a mutp53-R273H expression construct or empty pCDNA3 vector as a control. In addition, we performed gene expression analysis of U-251 MG cells transiently transfected with p53-specific siRNA or control siRNA (3 biological replicates each).
Project description:Med1 overexpression leads to induction of a wide spectrum of genes. Adenovirally-driven overexpression of Med1 in mouse liver stimulates hepatocyte DNA synthesis with enhanced expression of DNA replication, cell cycle control and liver specific genes as observed at day 3 and day 5 post injection. Med1 gene is amplified in a number of cancers so in this study we tested the hypothesis that Med1 by itself has the capacity to induce cell proliferation. Analysis of the Med1- induced gene expression showed a robust induction of a wide spectrum of genes involved in hepatocellular proliferation. Med1 floxed mice (Med1fl/fl) were injected with Ad-His-Med1 (adenovirus expressing Med1 gene) via tail vein and killed 3 or 5 days after injection. Ad-LacZ (adenovirus expression beta-galactosidase gene) injected mouse liver served as control. Total RNA were isolated from the liver and subjected to the microarray.
Project description:We describe a synthetic interaction screen to identify genes preferentially required for proliferation of p53-deficient human cancer cells. An isogenic pair of p53+/+ and p53-/- human colorectal HCT116 cell lines was stably transduced with a short hairpin RNA (shRNA) library encoded by lentiviruses that integrate in the genomic DNA of the host. Comparison of the frequency of each type of integrated lentivirus in the 2 cell types at an early time point (40hrs) and after about 12 mitotic cycles (10days), reveals genes whose knockdown preferentially impaired growth of p53-/- or p53+/+ HCT116 cells. Keywords: Functional Genomics The region of the integrated lentiviruses is PCR amplified from genomic DNA of the two transduced cell populations at an early and a late time point.