Project description:Classical NF-κB activity can be inhibited by overexpression of the IκBα super repressor (SR). To determine the role of NF-κB in rhabdomyosarcoma cells, we overexpressed the IκBα SR in RH30 rhabdomyosarcoma cells. IκBα SR was overexpressed in RH30 cells. RH30 vector cells were used as control group.
Project description:Classical NF-κB activity can be inhibited by overexpression of the IκBα super repressor (SR). To determine the role of MyoD in rhabdomyosarcoma cells with NF-kB inhibited, ablated MyoD expression with CRISPR/cas9 editing and clonal selection of RH30-SR cell lines to compare differences in expression following deletion
Project description:We used human Affymetrix microarrays to identify the up- or down-regulated gene expressions from MDA-MB-231 cells infected with control vector or Flag-SR-IkBa Experiment Overall Design: The pattern of gene expression from MDA-MB-231 cells transduced with retroviruses were analysed by RNA extraction and hybridization on Affymetrix microarrays. From expression profiles, we identified NF-kB target genes, and uncovered a novel and specific role of a gene in osteolytic bone metastasis.
Project description:The effect of the Smac mimetic BV6 on the transcriptional regulation in the alveolar rhabdomyosarcoma cell line RH30 was investigated by bulk RNA-sequencing. To this end, RH30 cells were treated with 5 µM BV6 for 24 h, or left untreated. Here, a regulation of several NF-κB target genes could be observed.
Project description:p73 is a p53 family transcription factor that plays critical roles during development and tumor suppression. We analyzed p73 activity using a combination of ChIP-on-Chip and gene expression profiling, both at baseline and after treatment with the mTOR inhibitor rapamycin. We generated an mTOR-p73 gene signature that predicts rhabdomyosarcoma tumor subtype and patient outcome, and is enriched for p73 target genes involved in mesenchymal stem cell differentiation and tumorigenesis. Rh30 rhabdomyosarcoma cells were infected with lentivirus (either control or expressing one of two RNAi constructs targeting p73) for 3 d, and treated with vehicle or 40 nM rapamycin for 24 h, and then total RNA was harvested. Experiments were performed in duplicate for a total of 8 samples. For p73 RNAi, a different targeting construct was used for each replicate.
Project description:ChIP-seq analysis of SNAIL binding sites in RH30 cells was performed to discover novel SNAIL binding sites in rhabdmyosarcoma cells.
Project description:Inflammatory signals are key in development and cell differentiation but their orchestration with pluripotency and stemness signals is poorly understood. Our previous work identified a chromatin function of IκBα, the NF-κB inhibitor, that is crucial for differentiation in different types of somatic stem cells. Here we demonstrate that deficiency of IκBα imposes a profound chromatin rewiring defect that impacts on DNA methylation, histone post-translational modifications and transcriptional regulation, stabilizing mouse embryonic stem cells (ESCs) in a ground state of pluripotency while preventing them from pluripotency exit and differentiation. By engineering separation-of-function mutants of IκBα with specific binding to either NF-κB or histones, we demonstrate that regulation of pluripotency state by IκBα is independent of NF-kB but requires the chromatin-related IκBα function.
Project description:Inflammatory signals are key in development and cell differentiation but their orchestration with pluripotency and stemness signals is poorly understood. Our previous work identified a chromatin function of IκBα, the NF-κB inhibitor, that is crucial for differentiation in different types of somatic stem cells. Here we demonstrate that deficiency of IκBα imposes a profound chromatin rewiring defect that impacts on DNA methylation, histone post-translational modifications and transcriptional regulation, stabilizing mouse embryonic stem cells (ESCs) in a ground state of pluripotency while preventing them from pluripotency exit and differentiation. By engineering separation-of-function mutants of IκBα with specific binding to either NF-κB or histones, we demonstrate that regulation of pluripotency state by IκBα is independent of NF-kB but requires the chromatin-related IκBα function.