Analysis of gene expression during Calcium induced differentiation of human primary keratinocytes (NHEK)
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE38628 | GEO | 2012/07/19
SECONDARY ACCESSION(S): PRJNA168295
REPOSITORIES: GEO
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Project description:Human primary keratinocytes were collected at 0, 1, 3, 6, 12, 24 and 48 hours after addition of 1.8mM Calcium and RNA was extracted. Primary normal human keratinocytes (NHEK) were collected and RNA was extracted 0, 1, 3, 6, 12, 24, and 48 after addition of 1.8mM Calcium.
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Project description:Human epidermal keratinocytes undergo tightly controlled program of cell differentiation, leading to the formation of cornified envelope. Primary keratinocytes in vitro, under calcium stimulation mimic the differentiation program observed in vivo. Analysis of the transcription profile of two cell population, such as proliferating cells and differentiating cells helps to discover new genes implicated in that process and to understand the mechanisms of regulation of the keratinocyte differentiation. Primary human keratinocytes were cultured under proliferating (Day 0, sub-confluent cells) and differentiating (seven days of high calcium medium) conditions. As a source of cells, we used normal skin from different body sites: back, foreskin, palmoplantar. RNA extracted from cultured primary human keratinocytes were isolated from five different donors. We compared the expression profiles of proliferating versus differentiating keratinocytes.
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Project description:Human primary keratinocytes were depleted of GRHL3 by siRNA and induced to differentiated for 2 days by addition of Calcium Primary normal human keratinocytes were transfected with GRHL3 or scrambled control siRNA using RNAi max (Life Technologies). 24 hours post transfection medium was raised to 1.8mM to induce differentiation. Cells were collected 48 hours later.
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Project description:Analysis of undifferentiated keratinocytes or differentiated keratinocytes stimulated with or without human cathelicidin antimicrobial peptide (CAMP) LL37. Results provide insight into the biological effects of CAMP on human keratinocytes. NHEKs were divided into two groups; low calcium (0.05 mM) and high calcium condition (1.6 mM). Then keratinocytes were stimulated with human cathelicidin antimicrobial peptide LL37 at 0, 2.56, and 7.68 M-NM-<M for 12 h to 24 h.
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Project description:To further investigate the question of cell-of-origin in keratinocyte cancer, an epigenetic clock was trained to learn and predict keratinocyte differentiation. Human EpSCs from skin biopsies were isolated and differentiation induced with calcium. Three replicates each were analyzed at the beginning of the experiment (0 h), as well as after 72 h and 96 h. The epigenetic clock trained on these replicates was further applied to single cell keratinocytes to predict their differentiation status.
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Project description:We have compared the response to TGFbeta1 in normal and v-rasHa transduced primary mouse keratinocytes using NCI cDNA microarrays. This analysis reveals that Ha-ras alters global TGFbeta1 mediated gene expression in a gene specific manner. The expression pattern of TGFbeta1 immediate early response genes and down regulation of cell cycle control genes is not altered by Ha-ras but the induction of most extracellular matrix genes is blocked. Using Smad3 null keratinocytes, we find that the majority of TGFbeta1 responsive genes in primary keratinocytes are Smad3 dependent, but patterns of transcriptional responses suggest that the ability of Ha-ras to block TGFbeta1 mediated gene expression is not dependent on Smad3. However, the combination of oncogenic ras and loss of Smad3 prevents the TGFbeta1 dependent suppression of genes associated with cell cycle progression and apoptosis observed with either genotype alone. In addition several extracellular matrix genes are super-repressed by TGFbeta1 in the Ha-ras Smad3 null keratinocytes. These data provide a genomic framework for understanding how disruptions of TGFbeta signaling and oncogenic ras cooperate to promote premalignant progression of primary keratinocytes. Keywords: time series design RNA from Balb/c primary and ras keratinocytes treated for 1, 6, 24, and 48 hours with TGFbeta1 at 1ng/ml was compared to RNA from the corresponding untreated controls. For each timepoint sample from primary and ras cells, multiple arrays (including dye swaps) were analyzed. There are 6, 7, 6, and 5 arrays analyzed for RNA from primary keratinocytes treated for 1, 6, 24, and 48 hours as compared to RNA from untreated primary cells, respectively. There are 6, 8, 7, and 5 arrays analyzed for RNA from ras keratinocytes treated for 1, 6, 24, and 48 hours as compared to RNA from untreated ras cells, respectively. There are 50 arrays analyzed for the Balb/c time-course dataset. RNA from Smad3 WT and KO primary and ras keratinocytes treated 48 hours with TGFbeta1 were compared to RNA from its untreated corresponding controls. There are 6 arrays analyzed for the Smad3 WT/KO data set. There are 56 arrays in this series.
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Project description:In this study we aim to determine the role of IL-4/STAT6 in gene expression in human keratinocytes using RNA-sequencing approach. Human keratinocytes were cultured for 2 or 5 days with calcium chloride to induce terminal differentiation as determined by the expression of epidermal differentiation complex genes. The cells were then stimulated with IL-4 for 3 and 24 hours, or along the 5 days culture period. We observed that IL-4 inhibits fully differentiation of keratinocytes, induces genes involved with production of inflammatory mediators, and reduces the healing capacity of human keratinocytes. Moreover, STAT6 controlled important genes involved with calcium binding, inflammation and epidermis development. Human keratinocytes were differentiated with calcium chloride for 2 days and incubated with media alone or 20ng/ml of recombinant human IL-4 for 3 and 24 hours. Human keratinocytes were differentiated with calcium chloride for 5 days with or wihout recombinant human IL-4 (20ng/ml). Keratinocytes transfected with control or STAT6 siRNA were differentiated with calcium chloride for 2 days and then stimulated with recombinant huma IL-4 for 24 hours.
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Project description:Cultured keratinocytes treated with TNFa in the presence or absence of NFkB inhibitor; time course 1, 4, 24 & 48 hrs. Keywords = Epidermis Keywords = skin Keywords = keratinocytes Keywords = inflammation Keywords = NFkB Keywords = TNFa Keywords: time-course
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