Expression analysis of OSK and Sall4+OSK reprogramming cells sorted for expression of miR-290 and/or miR-302.
Ontology highlight
ABSTRACT: MEFs were infected with Oct4, Sox2, Klf4 (+/- Sall4), sorted for miR-290/302 reporter expression at day 9 (OSK) or 12 (Sall4+OSK), and then profiled. Resulting iPSCs were also profiled. Mouse ES cells were differentiated and sorted for miR-290/302 reporter expression during Fgf/Activin differentiation (day 4 or day 7). Performed in biological triplicate. Biological triplicate samples represent three independent lines. Total RNA collected with Trizol. All processing conducted at the UCLA Neuroscience Genomics Core. MouseRef-8 v2.0 Expression BeadChips.
Project description:Wild-type or miR-302 knockout tissue was isolated from embryos during neurulation in replicate Performed in biological triplicate except for E7.5 WT which was in duplicate and E9.5 timepoints which were in quadruplicate. Biological replicate samples represent independent embryos. Total RNA collected with Trizol. All processing conducted at the UCLA Neuroscience Genomics Core. MouseRef-8 v2.0 Expression BeadChips.
Project description:Tumor-associated stromal cells can enable cancer cells to become insensitive to therapy. They can promote aggressive phenotype in cancer cells, which become less responsive to drugs such as BRAF inhibitors (BRAFi) used to treat melanomas. To clarify potential mechanism behind stromal influence on melanoma, we analyzed gene expression in Melmet 5 melanoma cells grown as mono-cultures or co-cultures with lung fibroblasts with/without BRAFi. We have shown that Melmet 5 growing as co-cultures gained a de-differentiated, invasive transcriptional state, which is known to be linked to BRAFi-resistance. The transcriptional changes induced by BRAFi were much larger in Melmet 5 mono-cultures compared to co-cultures, indicating a much dampened transcriptional response to BRAFi in melanoma under the influence of fibroblasts. We conclude that interaction with the stromal cells stimulate melanoma cell transition to the invasive de-differentiated phenotype, leading to a worse response to BRAF inhibitors. Total RNA was isolated from Melmet 5 cell line growing as mono- or co-cultures with fibroblasts for 72 hours and treated with BRAFi for the last 24 hours.
Project description:In vitro neural stem cell models are widely used to model a wide range of neuropsychiatric conditions. However, how well such models correspond to in vivo brain has not been evaluated in an unbiased, comprehensive manner. We used transcriptomic analyses to compare in vitro systems to developing human fetal brain and observed strong conservation of in vivo gene expression and network architecture in differentiating primary human neural progenitor cells (phNPCs). Conserved modules are enriched in genes associated with ASD, supporting the utility of phNPCs for studying neuropsychiatric disease. We also developed and validated a machine learning approach called CoNTExT that identifies the developmental maturity and regional identity of in vitro models. We observed strong differences between in vitro models, including hiPSC-derived neural progenitors from multiple laboratories. This work provides a framework for evaluating in vitro systems and supports their value in studying the molecular mechanisms of human neurodevelopmental disease. In this GEO submission, we upload data from 5 lines of phNPCs as well as hiPSCs cultured in two different laboratories all at multiple differentiation time points. phNPCs: For each of 5 lines generated from 3 donors (15-16 PCW), two independent differentiation experiments each containing two replicates were performed and harvested at four time points (1, 4, 8, 12 wks PD; ~16 samples per line; 77 total samples). We confirmed RNA integrity by RIN score with the Agilent 2100 Bioanalyzer (mean +/- sd: 9.16 +/- 0.78). iPSC: Two hiPSC datasets were RNA profiled as part of this study. hiPSCs grown in the Kosik lab was derived from two independent, non-isogenic IPS lines: one derived from a patient carrying a mutant Tau variant G55R and one reference control. For each of these lines, two samples were harvested at each of 0, 1, 4, and 8 weeks PD (total n=16 samples). hiPSCs grown in the Gage lab were from six samples derived from 3 control lines at each of two time points (0 and 4 wk PD, total n=12 samples). Samples were randomized to microarray chip by all biological variables of interest (donor, line, passage, replicate number, differentiation week, plate date, and RIN) to control for potential batch effects.
Project description:Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is a critical regulator of cell growth by integrating multiple signals (nutrients, growth factors, energy and stress) and is frequently deregulated in many types of cancer. We used a robust experimental paradigm involving the combination of two interventions, one genetic and one pharmacologic to identify genes regulated transcriptionally by mTORC1. In Tsc2+/+, but not Tsc2-/- immortalized mouse embryo fibroblasts (MEFs), serum deprivation downregulates mTORC1 activity. In Tsc2-/- cells, abnormal mTORC1 activity can be downregulated by treatment with rapamycin (sirolimus). By contrast, rapamycin has little effect on mTORC1 in Tsc2+/+ cells in which mTORC1 is already inhibited by low serum. Thus, under serum deprived conditions, mTORC1 activity is low in Tsc2+/+ cells (untreated or rapamycin treated), high in Tsc2-/- cells, but lowered by rapamycin; a pattern referred to as a M-bM-^@M-^\low/low/high/lowM-bM-^@M-^] or M-bM-^@M-^\LLHLM-bM-^@M-^]. We found that mTORC1 regulated the expression of, among other lysosomal genes, V-ATPases through the transcription factor EB (TFEB, Tcfeb in the mouse). The knockdown of Tfeb resulted in the 'flattening' of the LLHL pattern and allowed the identification of genes regulated by mTORC1 through Tfeb Mouse embryo fibroblasts (MEFs) wild type or deficient in Tsc2 expressing a Tfeb shRNA or scrambled shRNA vector were treated with 25 nM rapamycin or vehicle (methanol) for 24 h under low serum conditions (0.1% FBS)
Project description:Analysis of gene expression changes in differentiated human podocytes treated with the serum from patients with (DKD+) or without (DKD-) diabetic kidney disease when compared to normal subjects (C). The hypothesis is that the three groups can be distinghed by their differential gene expression pattern. The results obtained revealed important information regarding differences in gene expression in human podocytes treated with the serum from patients with (DKD+) or without (DKD-) diabetic kidney disease when compared to normal subjects (C). Human podocytes were contacted with the serum from patients with diabetes and kidney disease (DKD+) or without kidney disease (DKD-) and compared to normal human podocytes contacted with serum from patients without diabetes (C).
Project description:Analysis of gene expression changes in differentiated human podocytes treated with the serum from patients with (DKD+) or without (DKD-) diabetic kidney disease when compared to normal subjects (C). The hypothesis is that the three groups can be distinghed by their differential gene expression pattern. The results obtained revealed important information regarding differences in gene expression in human podocytes treated with the serum from patients with (DKD+) or without (DKD-) diabetic kidney disease when compared to normal subjects (C). Human podocytes were contacted with the serum from patients with diabetes and kidney disease (DKD+) or without kidney disease (DKD-) and compared to normal human podocytes contacted with serum from patients without diabetes (C).
Project description:Selective bromodomains inhibitors block the interaction between diverse bromodomains and extraterminal domains (BET) proteins and acetylated proteins. These inhibitors have shown beneficial effects in cancers malignancies and experimental inflammation in mouse models, but data on renal diseases are scarce. We have investigated the effect of the BET proteins inhibitor JQ1 in a mice model of unilateral ureteral obstruction. Treatment with JQ1 diminished renal damage, the presence of inflammatory cell infiltration and the upregulation of proinflammatory genes. The in vitro evaluation of JQ1 on TNF-α inducible genes in renal cells showed that BET inhibition modulated several biological processes, including inflammation or immune response. Moreover, gene-silencing experiments showed that BRD4 regulates several proinflammatory genes (IL-6, CCL-2 and CCL-5) and chromatin immunoprecipitation techniques demonstrated that BRD4 specifically binds to acetylated histone H3 in the promoter region of those genes. The nuclear factor-ï«B (NF-ï«B) pathway regulates renal inflammation. The RelA NF-ï«B subunit is activated by acetylation of lysine 310. In damaged kidneys and in TNF-α-treated renal cells, JQ1 blocked the nuclear translocation of RelA/NF-ï«B and NF-ï«B-mediated gene expression. Additionally, obstructed kidneys showed an activation of the Th17 immune response, which was diminished by JQ1 treatment. Our results demonstrate that the BET inhibition decreases renal inflammation by 3 independent mechanisms: 1) chromatin remodelling in the promoter regions of specific genes, 2) blocking NF-ï«B pathway activation, and 3) modulating the Th17 immune response. These results suggest that BET inhibitors could have important therapeutic applications in inflammatory renal diseases. HK2 cell line was treated with JQ1 or its enantiomer JQ1(-) (500nM) for 1 hour before stimulation or not with TNF-α (5ng/ml) for additional 3h. We analized the genes up- and down-regulated by TNF-α vs unstimulated cells, and further the genes downregulated or unaffected by JQ1 compared to the cells treated with the enantiomer (-) JQ1.