Microfluidic 3D HepG2 cell culture: Reproducing hepatic tumor gene and protein expression in in vitro scaffolds

2015 ◽  
Vol 15 (3) ◽  
pp. 340-350 ◽  
Author(s):  
Uta Fernekorn ◽  
Jörg Hampl ◽  
Frank Weise ◽  
Maren Klett ◽  
Annette Läffert ◽  
...  
Keyword(s):  
Cell Culture ◽  
Protein Expression ◽  
Hepg2 Cell ◽  
Hepatic Tumor ◽  
Gene And Protein Expression ◽  
Tumor Gene

scholarly journals Identification of pluripotent cells in bovine uterus: in situ and in vitro studies

Reproduction ◽  
2015 ◽  
Vol 149 (4) ◽  
pp. 317-327 ◽  
Author(s):  
Martyna Łupicka ◽  
Gabriel Bodek ◽  
Nahum Shpigel ◽  
Ehud Elnekave ◽  
Anna J Korzekwa
Keyword(s):  
Flow Cytometry ◽  
Protein Expression ◽  
Mrna Expression ◽  
Uterine Horn ◽  
Uterine Tissue ◽  
Pluripotent Cells ◽  
Flow Cytometry Assay ◽  
Myometrial Cells ◽  
Gene And Protein Expression

The aim of this study was to identify uterine pluripotent cells both in bovine uterine tissues as well in epithelial, stromal, and myometrial uterine cell populations. Moreover, the relationship of pluripotent markers expression with age and the uterine horn side was considered. Uterine tissue was collected from ipsilateral and contralateral horns (days 8–10 of the estrous cycle). Immunohistostaining for C-KIT, OCT3/4, NANOG, and SOX2 in uterine tissue was determined. mRNA expression of C-KIT, OCT3/4, NANOG and SOX2 was evaluated in uterine tissue relative to the age of the cow and uterine horn side. Gene and protein expression of these markers in the uterine luminal epithelial, stromal, and myometrial cells was evaluated by real-time PCR and western blotting respectively. The expression of pluripotent cell markers OCT3/4, NANOG, and SOX2 was identified by flow cytometry assay in epithelial, stromal, and myometrial cells. Multilineage differentiation of the bovine uterine cells was performed. mRNA expression of OCT3/4, NANOG, and SOX2 in uterine tissue was higher in the ipsilateral horn than in the contralateral horn. Flow cytometry assay revealed positive fluorescence for OCT3/4, NANOG, and SOX2 in all uterine cell types. Results showed the age-dependent expression of pluripotent markers in uterine tissue. Beside, the different expression of pluripotent cells in each horn of uterus suggests the influence of ovarian hormones on these characteristics. The highest mRNA and protein expression for pluripotent markers was observed in stromal cells among uterine cells, which indicates this population of cells as the main site of pluripotent cells in the cow uterus.

scholarly journals Cell signaling pathways in human mutant PAX6 corneal cells: an in vitro model for aniridia-related keratopathy

2021 ◽  
Author(s):  
Marta Słoniecka ◽  
André Vicente ◽  
Berit Byström ◽  
Fátima Pedrosa Domellöf
Keyword(s):  
Protein Expression ◽  
Signaling Pathways ◽  
Cell Fate ◽  
Expression Patterns ◽  
Pax6 Gene ◽  
Cas9 Nuclease ◽  
Extracellular Matrix Components ◽  
Gene And Protein Expression ◽  
Human Keratocytes

ABSTRACTPURPOSETo establish an in vitro model of aniridia-related keratopathy (ARK) using CRISPR/Cas9 engineered human keratocytes with mutations in the PAX6 gene, and to study the Notch Homolog 1, Translocation-Associated (Notch1), sonic hedgehog (SHH), mammalian target of rapamycin (mTOR), and Wnt/β-catenin signaling pathways in the PAX6 mutant keratocytes.METHODSPrimary human keratocytes were isolated from healthy corneas. Keratocytes were transduced with Cas9 lentiviral particles in order to create cells stably expressing Cas9 nuclease. Lentiviral particles carrying PAX6 sgRNA were transduced into the Cas9 keratocytes creating mutants. Analysis of signaling pathways was assessed by RT-qPCR for gene expression and western blot for protein expression.RESULTSHuman keratocytes stably expressing Cas9 nuclease were created. Keratocytes carrying PAX6 gene mutation were successfully generated. PAX6 mutant keratocytes showed modified expression patterns of extracellular matrix components such as collagens and fibrotic markers. Analysis of the Notch1, SHH, mTOR, and Wnt/β-catenin signaling pathways in the PAX6 mutant keratocytes revealed altered gene and protein expression of the key players involved in these pathways.CONCLUSIONSA properly functioning PAX6 gene in keratocytes is crucial for the regulation of signaling pathways important for cell fate determination, proliferation, and inflammation. Pax6 mutation in the in vitro settings leads to changes in these pathways which resemble those found in corneas of patients with ARK.

scholarly journals Uremia Impacts VE-Cadherin and ZO-1 Expression in Human Endothelial Cell-to-Cell Junctions

Toxins ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 404 ◽  
Author(s):  
Rayana Maciel ◽  
Regiane Cunha ◽  
Valentina Busato ◽  
Célia Franco ◽  
Paulo Gregório ◽  
...  
Keyword(s):  
Protein Expression ◽  
Intercellular Junctions ◽  
Serum Levels ◽  
Endothelial Damage ◽  
Cell Junctions ◽  
Cell Junction ◽  
Uremic Serum ◽  
Gene And Protein Expression ◽  
The Impact

Endothelial dysfunction in uremia can result in cell-to-cell junction loss and increased permeability, contributing to cardiovascular diseases (CVD) development. This study evaluated the impact of the uremic milieu on endothelial morphology and cell junction’s proteins. We evaluated (i) serum levels of inflammatory biomarkers in a cohort of chronic kidney disease (CKD) patients and the expression of VE-cadherin and Zonula Occludens-1 (ZO-1) junction proteins on endothelial cells (ECs) of arteries removed from CKD patients during renal transplant; (ii) ECs morphology in vitro under different uremic conditions, and (iii) the impact of uremic toxins p-cresyl sulfate (PCS), indoxyl sulfate (IS), and inorganic phosphate (Pi) as well as of total uremic serum on VE-cadherin and ZO-1 gene and protein expression in cultured ECs. We found that the uremic arteries had lost their intact and continuous endothelial morphology, with a reduction in VE-cadherin and ZO-1 expression. In cultured ECs, both VE-cadherin and ZO-1 protein expression decreased, mainly after exposure to Pi and uremic serum groups. VE-cadherin mRNA expression was reduced while ZO-1 was increased after exposure to PCS, IS, Pi, and uremic serum. Our findings show that uremia alters cell-to-cell junctions leading to an increased endothelial damage. This gives a new perspective regarding the pathophysiological role of uremia in intercellular junctions and opens new avenues to improve cardiovascular outcomes in CKD patients.

scholarly journals Adiponectin Expression in the Porcine Ovary during the Oestrous Cycle and Its Effect on Ovarian Steroidogenesis

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Anna Maleszka ◽  
Nina Smolinska ◽  
Anna Nitkiewicz ◽  
Marta Kiezun ◽  
Katarzyna Chojnowska ◽  
...  
Keyword(s):  
Protein Expression ◽  
Energy Homeostasis ◽  
Oestrous Cycle ◽  
Adiponectin Receptors ◽  
Basal Secretion ◽  
Ovarian Steroidogenesis ◽  
Gene And Protein Expression ◽  
Adiponectin Mrna ◽  
Theca Interna

Adiponectin is an adipose-secreted hormone that regulates energy homeostasis and is also involved in the control of the reproductive system. The goal of the present study was to investigate changes in adiponectin gene and protein expression in porcine ovarian structures during the oestrous cycle and to examine the effects ofin vitroadministration of adiponectin on basal and gonadotrophin- and/or insulin-induced secretion of ovarian steroid hormones. Both gene and protein expression of adiponectin were enhanced during the luteal phase of the cycle. Adiponectin affected basal secretion of progesterone by luteal cells, oestradiol by granulosa cells, and testosterone by theca interna cells. The gonadotrophin/insulin-induced release of progesterone from granulosa and theca interna cells and the release of oestradiol and androstenedione from theca cells was also modified by adiponectin. In conclusion, the presence of adiponectin mRNA and protein in the porcine ovary coupled with our previous results indicating adiponectin receptors expression suggest that adiponectin may locally affect ovarian functions. The changes in adiponectin expression throughout the oestrous cycle seem to be dependent on the hormonal status of pigs related to the stage of the oestrous cycle. The effect of adiponectin on ovarian steroidogenesis suggests that this adipokine influences reproductive functions in pigs.

The Histone Deacetylase (HDAC) Inhibitor Entinostat (SNDX-275) Targets Hodgkin Lymphoma through a Dual Mechanism of Immune Modulation and Apoptosis Induction.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1562-1562 ◽  
Author(s):  
Noor M Khaskhely ◽  
Daniela Buglio ◽  
Jessica Shafer ◽  
Catherine M. Bollard ◽  
Anas Younes
Keyword(s):  
Immune Response ◽  
Cell Death ◽  
Protein Expression ◽  
Cell Lines ◽  
Dependent Manner ◽  
Chemokine Production ◽  
Apoptosis Pathway ◽  
Gene And Protein Expression ◽  
Combination Studies

Abstract Abstract 1562 Poster Board I-585 Purpose SNDX-275 is an oral, class 1 isoform selective HDACi. Phase 1 studies in leukemia demonstrated the agent has a long half-life and that weekly or every other week dosing is sufficient for antitumor activity. Based on recent favorable in vitro and in vivo activity of several HDAC inhibitors in HL, we investigated the in vitro activity of SNDX275 in HL-derived cell lines. Methods For apoptosis and gene expression analysis 05 × 106 cells were incubated with 0.1-2 μM of SNDX-275 for 24-72 hours before they were examined for proliferation and cell death by the MTS assay and the annexin-PI and FACS analysis. For combination studies, cells were incubated with 0.1-2 uM of SNDX-275 and 1-20 nM of either gemcitabine or bortezomib for 48-72 hours. Gene and protein expression were measured by RT-PCR, western blot, and immunohistochemistry. SNDX-275 effects on a panel of 30 cytokines and chemokines was assayed on 05 × 106 cells after incubation of 48 hrs using a multiplex assay. Results SNDX-275 induced cell death in a dose and time dependent manner with an IC50 of 0.4 μM. At the molecular level, SNDX-275 increased H3 acetylation, up-regulated p21 protein expression, and activated the intrinsic apoptosis pathway by down-regulating the anti-apoptotic X-linked inhibitor or apoptosis (XIAP) protein, which was associated with activation of caspase 9 and 3. Combination studies demonstrated that SNDX-275 had synergistic effects when combined with gemcitabine and bortezomib. To further investigate the potential for SNDX-275 activity in HL we measured the effect of SNDX-275 on pathways that may contribute to an anti-tumor immune response. Dysregulated cytokine/chemokine production has been shown to contribute to HL pathology, including immune tolerance of the cancer cells. SNDX-275 increased IL12 p40-70, IP10, and RANTES, and decreased the level of IL13 and IL4, thus favoring Th1-type cytokines/chemokines. In addition, recent data has demonstrated that a variety of epigenetic-modulating drugs may up-regulate the expression of cancer testis tumor associated antigens, leading to a favorable immune response. None of the lines expressed the CTAs without induction. SNDX275 was able to induce CTA expression of SSX2 in L428 but not HDLM2 whereas MAGE-A was induced in both HL cell lines. NY-ESO expression was not induced. Conclusions Our studies demonstrate that SNDS-275 has dual effect on apoptotic and immunomodulatory pathways in HL. Furthermore, this data demonstrates that SNDX-275 may upregulate CTAs suggesting that this treatment may render the tumor more immunogeneic and susceptible to immune mediated killing with tumor-specific cytotoxic T lymphocytes. The selectivity profile of SNDX-275 also suggests that HDAC1 and 2 are the primary targets for HDAC inhibition in these cells. Phase 2 studies with SNDX-275 in HL are ongoing. Disclosures Younes: MethylGene: Honoraria, Research Funding.

scholarly journals Interleukin-1ß is a positive regulator of TIARP/STAMP2 gene and protein expression in adipocytes in vitro

FEBS Letters ◽  
2009 ◽  
Vol 583 (7) ◽  
pp. 1196-1200 ◽  
Author(s):  
Susan Kralisch ◽  
Grit Sommer ◽  
Sebastian Weise ◽  
Jana Lipfert ◽  
Ulrike Lossner ◽  
...  
Keyword(s):  
Protein Expression ◽  
Positive Regulator ◽  
Gene And Protein Expression

Endothelial cells respond to hyperglycemia by increasing the LPL transporter GPIHBP1

2014 ◽  
Vol 306 (11) ◽  
pp. E1274-E1283 ◽  
Author(s):  
Amy Pei-Ling Chiu ◽  
Fulong Wang ◽  
Nathaniel Lal ◽  
Ying Wang ◽  
Dahai Zhang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Expression ◽  
Binding Sites ◽  
Therapeutic Strategy ◽  
Density Lipoprotein ◽  
Streptozotocin Diabetes ◽  
Gene And Protein Expression ◽  
Lipoprotein Binding ◽  
Rate Limiting

In diabetes, when glucose uptake and oxidation are impaired, the heart is compelled to use fatty acid (FA) almost exclusively for ATP. The vascular content of lipoprotein lipase (LPL), the rate-limiting enzyme that determines circulating triglyceride clearance, is largely responsible for this FA delivery and increases following diabetes. Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein [GPIHBP1; a protein expressed abundantly in the heart in endothelial cells (EC)] collects LPL from the interstitial space and transfers it across ECs onto the luminal binding sites of these cells, where the enzyme is functional. We tested whether ECs respond to hyperglycemia by increasing GPIHBP1. Streptozotocin diabetes increased cardiac LPL activity and GPIHBP1 gene and protein expression. The increased LPL and GPIHBP1 were located at the capillary lumen. In vitro, passaging EC caused a loss of GPIHBP1, which could be induced on exposure to increasing concentrations of glucose. The high-glucose-induced GPIHBP1 increased LPL shuttling across EC monolayers. GPIHBP1 expression was linked to the EC content of heparanase. Moreover, active heparanase increased GPIHBP1 gene and protein expression. Both ECs and myocyte heparan sulfate proteoglycan-bound platelet-derived growth factor (PDGF) released by heparanase caused augmentation of GPIHBP1. Overall, our data suggest that this protein “ensemble” (heparanase-PDGF-GPIHBP1) cooperates in the diabetic heart to regulate FA delivery and utilization by the cardiomyocytes. Interrupting this axis may be a novel therapeutic strategy to restore metabolic equilibrium, curb lipotoxicity, and help prevent or delay heart dysfunction that is characteristic of diabetes.

scholarly journals Reanalysis of MERS, SARS and COVID-19 Infection Datasets using VirOmics Playground Reveals Common Patterns in Gene and Protein Expression

2020 ◽  
Author(s):  
Axel Martinelli ◽  
Murodzhon Akhmedov ◽  
Ivo Kwee
Keyword(s):  
Protein Expression ◽  
Bioinformatic Analysis ◽  
Data Sets ◽  
The Public ◽  
The Past ◽  
Genome Wide ◽  
Gene And Protein Expression ◽  
Heterogeneous Nature ◽  
User Friendly

AbstractThree lethal lower respiratory tract coronavirus epidemics have occurred over the past 20 years. This coincided with major developments in genome-wide gene and protein expression analysis, resulting in a wealth of datasets in the public domain. Seven such in vitro studies were selected for comparative bioinformatic analysis through the VirOmics Playground, a user-friendly visualisation and exploration platform we recently developed. Despite the heterogeneous nature of the data sets, several commonalities could be observed across studies and species. Differences, on the other hand, reflected not only variations between species, but also other experimental variables, such as cell lines used for the experiments, infection protocols and potential discrepancies between transcriptome and proteome data. The results presented here are available online and can be replicated through the VirOmics Playground.

scholarly journals Evaluation of Antioxidant and Antidiabetic Potential of Polyherbal Drug Made with Three Herbs: An In vitro Cell Culture Model

2021 ◽  
Vol 14 (4) ◽  
pp. 1627-1635
Author(s):  
P. Chandrasekaran
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Hepg2 Cell ◽  
Plant Extracts ◽  
Antioxidant Properties ◽  
Protein Glycation ◽  
Gas Chromatography Mass Spectrometry ◽  
Individual Plant ◽  
Hepg2 Cell Line

In diabetes, the postprandial phase is characterized by a rapid and large increase in blood glucose levels, and the possibility that the postprandial “hyperglycemic spikes” may be relevant to the onset of cardiovascular complications has recently received much attention. Medicinal use of herbal medicine in the treatment and prevention of diseases including diabetes has a long history compared to conventional medicine. These plants have no side effects and many existing medicines are derived from the plants. Hence, the current investigation was planned to make a poly herbal drug (PHD) through Punica granatum (fruits), Illicium verum (flowers) and Nyctanthes arbor (leaves) and assess their antioxidant and antidiabetic activities in vitro and in HepG2 cell line. The respective plant methanolic extracts and PHD are exposed to phytochemical assessment and to discriminate the bioactive factors by Gas Chromatography–Mass Spectrometry. We evaluated the antioxidant properties 2, 2-diphenyl-1-picrylhydrazyl scavenging, hydrogen peroxide scavenging, thiobarbituric acid reactive substances and total antioxidant activity of individual plant extracts and the PHD. At the same time, In vitro and cell culture approaches were used to assess the anti-diabetic activity. The PHD has a higher concentration of secondary metabolites than individual plant extracts, according to our findings. On the other hand, we also notice that PHD demonstrated higher antioxidant capability and considerable in vitro glucose-lowering effects along with noteworthy inhibition of ɑ-amylase, glucosidase, lipase, dipeptidyl peptidase-IV, collagenase and protein glycation in HepG2 cell line. In conclusion, this study clearly demonstrated the significant antioxidant and antidiabetic activities of the PHD. Hence, PHD may be used as a potential source in the management of diabetes, hyperglycemia and the related state of oxidative stress.

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