Mors lacking the chromosome 11 amplification, or normal lactating mammary glands from FVB/N mice. Genes

Mors lacking the chromosome 11 amplification, or normal lactating mammary glands from FVB/N mice. Genes labeled in red are expressed at higher than median levels and genes labeled in green are expressed at lower than median levels. Genes selected for further validation are indicated on the left sideBexpress high constitutive levels of c-Myc and contain the chromosome 11 amplification. The limitation of this model is that one cannot conclude that cell death is Myc-dependent or whether the tested genes have a more general effect on cell viability. To clarify this question, we Cynaroside chemical information established another model using normal murine mammary gland (NMuMG) epithelial cells with inducible expression of MycERTM where c-Myc is fused in frame with the mutated estrogen receptor-binding domain which makes it refractory to beta-estradiol but that can be activated by the addition of 4-hydroxytamoxifen [24]. First, we tested five different small hairpin RNA (shRNA) constructs for each gene and selected those that provided at least a 50 reduction in gene expression. These shRNAs were then stably expressed in Myc83 cells and NMuMG-MycERTM cellsFig. 3 Analysis of cell death induced by glucose deprivation or etoposide treatment in cells with JMJD6 knock-down. a Myc83derived cell lines (parental line from a MMTV-Myc tumor) with stable expression of two independent shRNAs targeting JMJD6 or with empty vector (EV) control were treated with 100 M etoposide or grown in glucose-free media for 20 h. Cell death was measured using CytoTox-Glo reagent. The experiments were repeated 3 times and the percentage of dead cells in each experiment was expressed relative to control (EV) cells. b NMuMG cells with MycERTM and shJMJD6 expression were first treated with 150 nM 4-OHT (MycON) or ethanol (MycOFF) for 24 h to activate Myc and then treated as in a Black bars–MycOFF (ethanol-treated cells), open bars–MycON (4OHT-treated cells). Results were normalized to EV control with MycOFF. *p < 0.05, **p < 0.Aprelikova et al. Clinical Epigenetics (2016) 8:Page 5 ofmodel system using NMuMG cells with constitutive overexpression of JMJD6 and an inducible c-Myc. As expected, ectopically expressed JMJD6 was localized to cell nuclei whereas control cells exhibited ectopic cytoplasmic expression of LacZ (Additional file 1: Figure S5A). The overall levels of JMJD6 in transfected cells were close to physiological levels observed in cells with amplified chromosome 11 (about 3-fold over control cells, Additional file 1: Figure S5B). Cells expressing high levels of c-Myc proved to be sensitive to multiple stress conditions, including depletion of nutrients or growth factors, or treatment with DNA-damaging agents, leading to cell death. C-Myc induction in NMuMG cells followed by exposure to four different stress conditions resulted in a significant increase in cell death, which was reduced by co-expression with JMJD6 (Fig. 4a ).JMJD6 is an iron- and 2-oxoglutarate-dependent dioxygenase with the capability to hydroxylate lysine residues PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28945807 in histone and non-histone proteins [26, 29, 30]. Lysine hydroxylation of RNA splicing factors results in production of differentially spliced pre-messenger RNA (mRNA) molecules [31], while hydroxylation of histone proteins may result in changes in transcriptional regulation of targeted gene expression [32]. In order to determine whether the enzymatic activity of JMJD6 is necessary for the inhibition of Myc-induced cell death, we mutated His187 to Ala in the iron-.