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Real-time quantitative PCR analysis of human EBV-B cells during UPR

ABSTRACT: Human B cells were isolated from peripheral blood from one healthy donor and two patients with Common Variable Immunodeficiency (CVID) and were immortalized by infection with Epstein Barr virus (EBV) in vitro. Immortalized EBV-B cells (in biological triplicates) were treated twelve different ways for 4 hours: 1.untreated control, 2. Thapsigargin (Tg) 1 µM, 3. Tunicamycin (Tm) 10 µg/ml, 4. 4-phenylbutyrate (4-PBA) 1 mM, 5. Tg 1 µM plus 4-PBA 1 mM, 6. Tm 10 µg/ml plus 4-PBA 1 mM, 7. Dimethyl sulfoxide (DMSO) 1 mM, 8. Tg 1 µM plus DMSO 1 mM, 9. Tm 10 µg/ml plus DMSO 1 mM, 10. Tauroursodeoxycholic acid (TUDCA) 5 mM, 11. Tg 1 µM plus TUDCA 5 mM, and 12. Tm 10 µg/ml plus TUDCA 5 mM. We used Applied Biosystems TaqMan OpenArray Real-Time PCR System (Thermo Scientific) to quantitate gene expression of relevant genes from the cells (in technical triplicates).

ORGANISM(S): Homo sapiens

PROVIDER: GSE126201 | GEO | 2020/01/19

REPOSITORIES: GEO

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Xenopus laevis Cdc45-FLAG interactions on chromatin during DNA replication

Project description:The aim of the project was to purify Xenopus replisome from replicationg chromatin assembled in Xenopus laevis egg extract. To this end recombinant Cdc45-TEV-His10-FLAG5 was expressed in bacteria and purified. 4 ml of Xenopus laevis egg extract was activated into interphase and supplemented with 10 ng/µl of demembranated sperm DNA, 70 nM recombinant Cdc45, 40 µM aphidicolin, 5 mM caffeine and incubated at 23°C for 60 min. Chromatin was isolated in ANIB100 buffer (50 mM HEPES pH 7.6, 100 mM KOAc, 10 mM MgOAc, 2.5 mM Mg-ATP, 0.5 mM spermidine, 0.3 mM spermine, 1 µg/ml of each aprotinin, leupeptin and pepstatin, 25 mM β-glycerophosphate, 0.1 mM Na3VO4 and 10 mM 2-chloroacetamide) as described previously (Gillespie, Gambus et al. 2012). Chromatin pellets re-suspended in ANIB100 containing 20% sucrose. Protein complexes were released from chromatin by digestion with 4 U/µl benzonase nuclease (E1014-25KU, Sigma) and sonicated for 5 min using a sonicator with settings: 30 sec on, 30 sec off, low power (Bioruptor Diagenode UCD-200). Immunoprecipitation was performed using 100 µl of anti-FLAG M2 magnetic beads (Sigma-Aldrich). Before elution the sample was washed four times with 1 ml of ANIB100 20% sucrose, ANIB100 20% sucrose 0.1% Triton X-100, ANIB100 and elution buffer (25 mM HEPES pH 7.5, 100 mM KAc, 5 mM MgAc, 1 mM ATP and 0.02% NP-40), respectively. The sample was eluted adding 250 µM 3xFLAG peptide (Stratech) to 200 µl of elution buffer and a small proportion of it analysed by mass spectrometry.

2023-10-27 | PXD044422 | Pride

Identification of interleukin-4 (IL-4) targets in chronic lymphocytic leukemia (CLL)

Project description:Gene expression profiles of CLL cells and normal B cells (NBC) treated for 18 hr with 10 ng/ml IL-4, compared with culture with nothing (Ctrl) and the basal (Pre) samples. Patient CLL01 was also treated with IL-4 plus 5 mg/ml ERK activation inhibitor peptide I, or IL-4 plus 10 µM NFkB activation inhibitor, and IL-4 plus 100 mg/ml cyclic-pifithrin-α

2014-10-06 | GSE55288 | GEO

Cardiomyocytes microarray

Project description:we employed DNA microarray platform to compare the gene expression patterns in primary human cardiomyocytes treated with trastuzumab (50µg/ml), trastuzumab (50µg/ml) plus pertuzumab (50µg/ml), T-DM1 (10 µg/ml), or control (no treatment).

2017-11-28 | GSE107385 | GEO

Real-time quantitative PCR analysis of Nrf2, HO-1, IL-6 and VEGF-A genes in human retinal pigment epithelium (RPE-19) cells

Project description:Human RPE-19 cells were treated with either Trolox (50 µg/mL), lutein (10 µg/mL) or gac peel extracts (0-200 µg/mL) for 12, 24, 48 or 72 h before being exposed to 500 µM H2O2 for 24 h prior to RNA collection. Results were presented as the fold change compared to untreated controls ± SEM (n=3), different letters indicated statistical significance at p≤0.05 between different treatments (Analysis by One-way ANOVA with Turkey’s LSD test).

2023-12-11 | GSE237892 | GEO

Microglia P2Y6 receptor signaling regulates a broad range of innate immune responses after TLR engagement

Project description:We analyzed the transcriptomes of adult primary rhesus macaque microglia that were exposed to 10 µg/mL uLPS, in the absence or presence of 5 µM P2RY6 antagonist MRS2578

2022-02-04 | GSE195866 | GEO

Cryptococcal H99 cells grown in 8 conditions for capsule induction

Project description:To understand the relationship between gene expression and capsule formation, H99 cells were cultured overnight at 37ºC in the following eight conditions: low iron medium with or without both 500 mM ethylenediaminetetraacetic acid (EDTA) and 10 mM bathophenanthroline disulfonate (BPDS); phosphate-buffered saline (PBS) with or without 10% v/v fetal bovine serum; Dulbecco's Modified Eagle's Medium (from Sigma) in ambient air or 5% CO2; and Littman's medium with either 0.01 µg/ml or 1 µg/ml thiamine.

2012-01-01 | GSE31911 | GEO

CRAC of yeast RNA polymerase II in WT and isw1Δ cells in standard conditions or upon ER stress.

Project description:We obtained RNA polymerase II occupancy profile across the genome of WT and isw1Δ Saccharomyces cerevisiae strains grown under normal conditions or after ER stress induction for 2H with1 µg/mL tunicamycin (Tm).

2022-10-06 | GSE207652 | GEO

in vitro differentiated Th0, Th17, and Tr1 cells

Project description:Gene expression profiling of in vitro differentiated murine Th cell subsets. Flow cytometrically sorted naive Th cells (CD4+ CD44- Foxp3-) were polyclonally stimulated in vitro for 3 days using 4 µg/ml plate-bound antibody to CD3 (145-2C11) and 2 µg/ml soluble antibody to CD28 (PV-1). Th0 cells were cultured in the absence of exogenous cytokines. Th17 cells were differentiated with 50 ng/ml IL-6 plus 0.5 ng/ml TGF-β. Tr-1 cells were differentiated with 100 ng/ml IL-27 plus 0.5 ng/ml TGF-β.

2014-03-20 | GSE56021 | GEO

Cell culture-based model system for FOXO3 activity

Project description:MCF7 and MDA-MB-231 breast cancer cell lines were cultured in DMEM-F12 containing 10% FBS (Lonza) 100U/ml penicillin and 100 µg/ml streptomycin (Lonza). Transfecting third generation packaging vectors using Poly-ethylenimine into HEK293T cells generated lentiviral particles (17). MCF7 and MDA-MB-231 cells were stably transduced with lentivirus containing pINDUCER20-FOXO3.A3, allowing doxycycline induced expression of constitutively active FOXO3 (FOXO3.A3). Cells were treated with 20% FBS or 10 µM PI3K inhibitor LY294002 (Selleckchem) for 16 hours to activate and inactivate the endogenous PI3K pathway, respectively. FOXO3.A3 expression was induced by 16 hours treatment with 10 ng/ml doxycycline.

2018-06-06 | GSE113479 | GEO

Effects of prostate cancer cells on osteoblasts

Project description:Primary murine osteoblasts were isolated form the calvariae of newborn mice. 10 days after the addition of ascorbic acid (50 µg/ml) and ß-glycerophosphate (10 mM), cells were serum-starved over night and then incubated for 6 hours with condtioned medium of MDA-PCa2b cells or conditioned medium of PC-3 cells Keywords: expression profiling by array

2010-07-01 | GSE17348 | GEO

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