Project description:We report the application of microarray sequencing technology for profiling of acetaminophen response in MDMXC462A mice. These data were used to evaluate differential gene expression and changes in cell composition using canonical markers for mouse liver cells.
Project description:We report the application of scRNA sequencing technology for profiling of acetaminophen response in MDMXC462A mice. These data were used to evaluate differential gene expression and changes in cell composition using canonical markers for mouse liver cells.
Project description:To investigate the effect of MDMX depletion on the transcriptome of MEL202 cells we established 5 derivatives either containing a doxycyline- inducible Contrl shRNA or with 4 distinct MDMX -targeting shRNAs In thee biological replicates the cells were treated with doxycycline for 48 hrs after which RNA was isolated
Project description:Genetic disruption of thioredoxin reductase 1 protects against acetaminophen (APAP) toxicity. To determine the role of the thioredoxin system on xenobiotic metabolism we challeneged wildtype and txnrd1liver-null mice with acetaminophen. Adult male wildtype and txnrd1 liver-null mice (C57BL6/J) were treated with either saline (PBS) or 100mg/kg APAP. Liver RNA was harvested eight hours after challenge and processed for microarray analysis. Comparison of 2 treatment conditions in 2 genotypes, biological replicates in triplicate.
Project description:The p53 inhibitor MDMX is overexpressed in the vast majority of patients with acute myeloid leukemia (AML). Utilizing hematopoietic stem cells from four non-leukemic/pre-leukemic murine models, we performed bulk transcriptomic analysis to evaluate the impact of Mdmx overexpression.
Project description:Gene expression profiles of sandwich-cultured primary human hepatocytes exposed to 5 mM and 10 mM acetaminophen were used in a parallelogram approach in order to compare gene expression responses between rat and human using in vitro cellular models, hepatocytes, and between rat in vitro and in vivo. Experiment Overall Design: Samples were retrieved from acetaminophen treated human hepatocyte cultures from 5 individuals (5 biological replicates). Experiment Overall Design: Human hepatocytes from each replicate were treated with 0, 5, and 10 mM acetaminophen for 24 h. Experiment Overall Design: This resulted in (3x5) 15 dual channel arrays on which control (0 mM acetaminophen) and reference samples (0, 5, and 10 mM acetaminophen) were hybridized.
Project description:The two murine double minute (MDM) family members, MDM2 and MDMX are a well-established negative regulator of p53 activity. Under DNA damage conditions, MDM2 and MDMX are phosphorylated near their RING domains (serine 395 at MDM2 and serine 403 at MDMX) and switch to act as p53 positive regulators. MDMX binds to TP53 mRNA and acts as a chaperone for RNA structure, enabling MDM2 to bind. This interaction enhances TP53 mRNA translation, leading to increased p53 protein production. While the biological significance of this interaction has been described, the specific features of the MDMX-RNA interaction remain poorly understood. We used various MDMX protein constructs to characterize binding to TP53 mRNA and identified the RING domain as a key element, modulated by the presence of other domains. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) and binding assays in high salt conditions demonstrate that the whole protein participates in RNA interaction, with the C-terminal domain likely providing the contact with RNA by electrostatic forces. Modulating pH to influence amino acid protonation showed that the full-length protein binding to RNA is more resilient to pH changes than its truncated C-terminus (322-490). We show that protein structural changes induced by the chelating agent EDTA or the reducing agent TCEP enhances RNA binding by promoting partial structural destabilization of the protein. Our findings suggest that the MDMX/TP53 mRNA interaction is complex, with the RING domain binding to RNA and being supported by the entire protein, which acts as a scaffold for the RNA interaction. These results contribute to a better understanding of MDMX´s role in TP53 mRNA binding and provide valuable insights for future investigation of the MDM2-MDMX-TP53 mRNA complex, which is crucial for p53 stabilization and activation under DNA-damaging conditions.