Deregulated Myc Requires MondoA for Metabolic Reprogramming and Tumorigenesis
ABSTRACT: In order to investigate the effect of MondoA loss on the expression of Myc-dependent genes, we performed a microarray analysis from RNAs isolated from TET21N cells expressing either control (siControl) or MondoA (siMondoA) siRNAs either with (NT) or without (Doxy) induced N-Myc expression. TET21N cells were grown in medium with either Doxy (Myc-Off) or No Treatment (Myc-On), then transiently transfected with either non-specific siRNA or MondoA siRNA in replicates. Cells were then lysed and RNA isolated.
Project description:In order to investigate the effect of MondoA loss on the expression of Myc-dependent genes, we performed a microarray analysis from RNAs isolated from TET21N cells expressing either control (siControl) or MondoA (siMondoA) siRNAs either with (NT) or without (Doxy) induced N-Myc expression. Overall design: TET21N cells were grown in medium with either Doxy (Myc-Off) or No Treatment (Myc-On), then transiently transfected with either non-specific siRNA or MondoA siRNA in replicates. Cells were then lysed and RNA isolated.
Project description:Ablation of ERRalpha significantly delays ERBB2-induced mammary tumorigenesis and ERRalpha regulates genes of the ERBB2 amplicon. To further investigate the relationship between ERRalpha activity and RTK signaling, we depleted ERRalpha in SKBr3 cells upon serum starvation, EGF treatment or heregulin treatment. Inhibition of ERBB2 signaling using the RTK inhibitor lapatinib impacts on ERRalpha stability. Since we have shown that the development of lapatinib-resistance restaures the expression of ERRalpha in breast cancer cells, we performed depletion of ERRalpha in SKBr3 cells that have developped resistance to lapatinib treatment in order to identify a potential reprogramming of ERRa transcriptional activity associated to lapatinib resistance, For the study of growth factor effec on ERRalpha activity, total RNA was obtained from human SKBr3 breast cancer cells cultured in DMEM deprived of FBS (starved) for 24 hours and treated with PBS, EGF (100uM) or Heregulin (100uM) for an additional 24h. Cells were transfected with siRNA against ERRalpha or with siControl for 60 hours prior to harvesting. For the effect of ERRalpha in lapatinib resistance cells, parental SKBr3 cells (pSKBr3) and Lapatinib-resistant cells (LRSKBr3, maintained in 2uM lapatinib) were transfected with siControl (siC) or siERRalpha for 60 hours prior to harvesting and RNA extraction
Project description:RNF31 is atypical E3 ligase, which is highly expressed in human breast cancers. In order to understand the effect of RNF31 depletion effect in breast cancers, we knocked down the expression of RNF31 in MCF-7 cells. We aim to find the significantly changed pathways and cell biology behavior. 100uM RNF31 siRNA was applied to transfect the MCF-7 cells, while 100uM siControl was used as the control. Cells were cultured in DMEM red medium with 10% of FBS. Seventy two hours of post transfection, the total RNA was extracted to perform RNA expression analysis.
Project description:Transcriptional profiling of the prostate adenocarcinoma cell line PC-3 comparing control cells transfected with a pool of oligonucleotides control (siControl) with cells transfected a pool of oligonucleotides specific to inhibit ARHGAP21 expression (siARHGAP21). The efficiency of the transfection was checked with quantitative PCR and western blotting. Goal was to determine the effects of ARHGAP21 silencing on global PC-3 gene expression. Two-condition experiment, siControl vs. siARHGAP21 PC-3 cells with three biological samples of PC3 cells transfected with a pool of oligonucleotides control (siControl 1, 2 and 3) and three biological samples of PC3 cells transfected with a pool of oligonucleotides specific to inhibit ARHGAP21 (siARHGAP21 1, 2 and 3). For each sample (three siControl and three siARHGAP21), two technical replicates were performed (labelled with Cy3 or Cy5).
Project description:Expression profiling after Klf4 KD during EMT in NMuMG reveals a significant number of genes that are transcriptionally deregulated Gene expression profiling is performed in Klf4 ablated (siKlf4) NMuMG cells. Cells transfected with siControl is used as a control. The cells were either treated with TGFβ (2ng/ml) or not.
Project description:Expression profiling after Sox4 knockdown (KD) during epithelial to mesenchymal transition (EMT) in NMuMG reveals a significant number of genes that are transcriptionally deregulated. Gene expression profiling is performed in Sox4-ablated (siSox4) NMuMG cells. Cells transfected with siControl is used as a control. The cells were either treated with transforming growth factor-beta (TGFβ; 2ng/ml) or not.
Project description:To investigate the role of YAP/TAZ as b-catenin inhibitors, we compared the expression profiles of Rex1GFPd2 ES cells transfected with siControl#1, siControl#2, siYAP/TAZ#1, siYAP/TAZ#2 and cultured in 2i medium or PD-only medium Overall design: We collected RNA from Rex1GFPd2 embryonic stem (ES) cells transfected with siControl#1, siControl#2, siYAP/TAZ#1, siYAP/TAZ#2. Cells were maintained in 2i medium (2i=PD0325901+CHIR99021) or switched to PD (PD0325901)-only medium after 24 hours from siRNA transfection. Cells were harvested 72 hours post-siRNA transfection. Samples were then processed for total RNA extraction and hybridization on Affymetrix microarrays.
Project description:Specific types of human papillomaviruses (HPVs) cause cervical cancer, the second most common tumor in females worldwide. Both cellular transformation and the maintenance of the oncogenic phenotype of HPV-positive tumor cells are linked to the expression of the viral E6 and E7 oncogenes. In order to identify downstream cellular target genes for the viral oncoproteins, we silenced endogenous E6 and E7 expression in HPV-positive HeLa cells by RNA interference (RNAi). Subsequently, we assessed changes of the cellular transcriptome by genome-wide microarray analysis. We identified, 648 genes wich were either downregulated (360 genes) or upregulated (288 genes) upon inhibition of E6/E7 expression. A large fraction of these genes is involved in tumour-relevant processes, such as apoptosis control, cell cycle regulation, or spindle formation. Others may represent novel cellular targets for the HPV oncogenes, such as a large group of C-MYC-associated genes involved in RNA splicing. Keywords: siRNA mediated knockdown Overall design: Hybridizations were performed on RZPD Unigene 3.1 cDNA microarrays (37.5K). Gene expression was analyzed by performing 6 microarray hybridizations, using three cell culture replicates treated with siRNA 18E7-3.5 or control siRNA siControl. Each replicate of the 18E7-3.5 experiments was hybridized against a replicate of the siControl experiment, including a technical dye swap.
Project description:We used mass spectrometry-based proteomics to unravel anaplastic lymphoma kinase (ALK) signaling in the ALK and MYCN amplified neuroblastoma cell line, NB1. We specifically measured the ALK phosphoproteome upon siRNA depletion of ALK and upon ALK inhibition using the ALK-targeting small-molecule inhibitor lorlatinib. For quantitative phosphoproteomics we used a tandem mass tag (TMT)-based approach. Conditions for the TMT 11-plex setup is specified below. For each siRNA depletion experiment, NB1 cells were treated with siRNA (80 nM; as specified below) for 48 hours prior to stimulation with 0.1% DMSO for 30 minutes. For inhibitor treatment, NB1 cells were treated for 30 minutes with either 10 microM or 10 nM lorlatinib. The experimental treatment conditions and TMT11-plex labeling are specified below: 126: siControl replicate 1, 0.1% DMSO 127N: siControl replicate 2, 0.1% DMSO 127C: siControl replicate 3, 0.1% DMSO 128N: siALK sequence 1, 0.1% DMSO 128C: siALK sequence 2, 0.1% DMSO 129N: siALK mix of sequence 1 and 2, 0.1% DMSO 129C: 10 microM lorlatinib replicate 1 130N: 10 microM lorlatinib replicate 2 130C: 10 microM lorlatinib replicate 3 131N: 10 nM lorlatinib replicate 1 131C: 10 nM lorlatinib replicate 2
Project description:Estrogen receptor α (ERα) is a nuclear receptor that is the driving transcription factor expressed in the majority of breast cancers. Recent studies have demonstrated that the liver receptor homolog-1 (LRH-1), another nuclear receptor, is ERα-regulated in breast cancer cells. Further, LRH-1 stimulates proliferation and promotes motility and invasion of breast cancer cells. To determine the mechanisms of LRH-1 action in breast cancer cells, we carried out gene expression microarray analysis following siRNA-mediated LRH-1 knockdown. Interestingly, gene ontology (GO) category enrichment analysis of the genes differentially regulated in the presence or absence of LRH-1 identified estrogen responsive genes as the most highly enriched GO categories. To further define LRH-1 target genes, we performed chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq) to identify genomic targets of LRH-1. Remarkably, ChIP-seq showed LRH-1 binding at many ERα binding sites. Analysis of select binding sites confirmed regulation of ERα-regulated genes by LRH-1 through binding to estrogen response elements, as exemplified by the TFF1/pS2 gene. Finally, LRH-1 over-expression stimulated ERα recruitment, whilst LRH-1 knockdown reduced ERα recruitment to ERα binding sites. Taken together, our findings establish a key role for LRH-1 in the regulation of ERα target genes in breast cancer cells and identify a mechanism in which co-operative binding of LRH-1 and ERα at estrogen response elements controls the expression of estrogen-responsive genes. MCF-7 cells were transfected with LRH-1 siRNA #2, #3, or with a non-targeting siRNA (siControl) for 72 hours. Following assessment of RNA integrity, four biological replicates for each siRNA treatment were used for microarray analysis.