Project description:Human m6A-mRNA&lncRNA Epitranscriptomic Microarray of arsenite-transformed human keratinocytes (HaCaT-T cells, 1 μM arsenite exposure for 50 passages) compared to its control HaCaT cells (passed for 50 passages without arsenic exposure).
Project description:The purpose of this study is to search for aberrant genes in HaCaT keratinocytes after chronic exposure to arsenic trioxide. The objective of the investigation was to discover the mechanism of arsenic carcinogenicity in human epidermal keratinocytes. We hypothesize that a combined strategy of DNA microarray, qRT-PCR and gene function annotation will identify aberrantly expressed genes in HaCaT keratinocyte cell line after chronic treatment with arsenic trioxide. HaCaT cells were chronically exposed to 0.5µg/mL arsenic trioxide (As2O3) up to 22 passages and RNA was extracted. Microarray data analysis identified 14 up-regulated genes and 21 down-regulated genes in response to arsenic trioxide
Project description:The purpose of this study is to search for aberrant genes in HaCaT keratinocytes after chronic exposure to arsenic trioxide. The objective of the investigation was to discover the mechanism of arsenic carcinogenicity in human epidermal keratinocytes. We hypothesize that a combined strategy of DNA microarray, qRT-PCR and gene function annotation will identify aberrantly expressed genes in HaCaT keratinocyte cell line after chronic treatment with arsenic trioxide. HaCaT cells were chronically exposed to 0.5M-BM-5g/mL arsenic trioxide (As2O3) up to 22 passages and RNA was extracted. Microarray data analysis identified 14 up-regulated genes and 21 down-regulated genes in response to arsenic trioxide Two experimental groups: 1. The treatment group was sub-cultured up to passage 22 to establish a chronic exposure state. 2. The passage control group was also sub-cultured up to 22 passages but with no exposure to arsenic trioxide. 4 technical replicates with 3 replicates making a total of 8X3 =24 samples HaCat Cell untreated (passage control): 1. H1_H001, H1_H002, H1_H003 2. H2_ H004, H2_H005, H2_H006 3. H3_ H007, H3_H008, H3_H009 4. H4_ H010, H4_H011, H4_H012 HaCat Cell treated with 0.5M-BM-5g/ml of arsenic trioxide: 5. A1_H013, A1_H014, A1_H015 6. A2_H016, A2_H017, A2_H018 7. A3_H019, A3_H020, A3_H021 8. A4_H022, A4_H023, A4_H024 Cell Type: Human Skin Keratinocyte: 1.5 M-CM-^W105 HaCaT cells were cultured in 7.5 ml of complete DMEM containing 10% Fetal Bovine Serum (FBS) and 1% penicillin, streptomycin in T-25 culture plate. Cells were incubated in a humidified atmosphere with 5% CO2 at 37 M-BM-:C. The treatment groups were exposed to 0.5M-BM-5g/mL As2O3 (equivalent to LC 0.5), and passaged at 90% confluent. Total RNA was extracted from 4 technical replicates of unexposed HaCaT cells and HaCaT cells chronically exposed to arsenic trioxide up to passage 22 using RNA STAT-60 (TEL-TEST, INC, Friendswood, TX, USA).
Project description:Modulation of signaling pathways upon chronic arsenic exposure remains poorly studied. Here we carried out SILAC-based quantitative phosphoproteomics analysis to dissect the signaling induced upon chronic arsenic exposure in human skin keratinocyte cell line, HaCaT. We identified 4,171 unique phosphosites derived from 2,000 proteins. We observed differential phosphorylation of 406 phosphosites (2-fold) corresponding to 305 proteins. Several pathways involved in cytoskeleton maintenance and organization were found to be significantly enriched (p<0.05). Our data revealed altered phosphorylation of proteins associated with adherens junction remodeling and actin polymerization. Kinases such as protein kinase C iota type (PRKCI), mitogen-activated protein kinase kinase kinase 1 (MAP3K1), tyrosine-protein kinase BAZ1B (BAZ1B) and STE20 like kinase (SLK) were found to be hyperphosphorylated. Our study provides novel insights into signaling perturbations associated with chronic arsenic exposure in human skin keratinocytes.
Project description:Immortalized keratinocytes HaCaT is popular model for skin research (toxicity, irritation, allergic reactions or interaction of cells). They maintain stable keratinocyte phenotype and respond to keratinocyte differentiation stimuli. However, the programs of stratification and expression of differentiation markers in HaCaT keratinocytes are aberrant. HaCaT cells bear two mutant p53 alleles (R282Q and H179Y) which contain Gain-of-Function (GOV) mutations acquired as a result of spontaneous immortalization (mutp53). At the same time, mutp53 acts as a transcription factor, and also affects interaction of p63 protein with its transcription targets. It is known that proteins of the P53 family play an important role in regulating processes of proliferation and differentiation of human keratinocytes.At the same time, the role of mutp53 in these processes is not fully clear. We present data sets obtained as a result of high-performance proteomic analysis of immortalized HaCaT keratinocytes with p53 knockout in two different states: sub-confluent and confluent, which are characterized by different intensity of cell differentiation processes. As a protocol for proteomic profiling of cells, we used the approach of obtaining LC-MS/MS measurements followed by their processing with Progenesis LC-MS software (Nonlinear Dynamics Ltd.)
Project description:We used RNA_Seq to quantify cellular transcripts in human spontaneously immortalized HaCaT keratinocytes upon knock out of GALNT1, GALNT2, or GALNT3 - most abundantly expressed enzyme isoforms initiating mucin-type O-linked glycosylation.
