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1.
ACS Omega ; 9(23): 25094-25105, 2024 Jun 11.
Article in English | MEDLINE | ID: mdl-38882105

ABSTRACT

Nonalcoholic fatty liver disease (NAFLD), which is a major cause of chronic liver disease, is characterized by fat accumulation in the liver. Existing models struggle to assess medication effects on liver function in the context of NAFLD's unique inflammatory environment. We address this by developing a 3D in vitro NAFLD model using HepG2 and THP-1 cells (mimicking liver and Kupffer cells) cocultured using transwell and hydrogel system. This mimics liver architecture and allows for manipulation of the immune environment. We demonstrate that the model recapitulates key NAFLD features: steatosis (induced by fatty acids), oxidative stress, inflammation, and impaired liver function embodying the interrelationship between NAFLD and the surrounding immune environment. This versatile model offers a valuable tool for preclinical NAFLD research by incorporating a disease-relevant immune environment.

2.
Tissue Eng Regen Med ; 20(7): 1173-1190, 2023 12.
Article in English | MEDLINE | ID: mdl-37843784

ABSTRACT

BACKGROUND: The emergence of various infectious diseases and the toxic effects of hyperinflammation by biotherapeutics have highlighted the need for in vitro preclinical models mimicking the human immune system. In vitro models studying the relationship between hyperinflammation and acute renal injury mainly rely on 2D culture systems, which have shown limitations in recapitulating kidney function. Herein, we developed an in vitro kidney toxicity model by co-culturing 3D engineered kidney proximal tubules cells (RPTEC/TERT1) with human peripheral blood mononuclear cells (PBMC). METHODS: RPTEC/TERT1 were sandwich cultured to form 3D renal tubules for 16 days. The tubules were then co-cultured with PBMC using transwell (0.4 µm pores) for 24 h. Hyperinflammation of PBMC was induced during co-culture using polyinosinic-polycytidylic acid (polyI:C) and lipopolysaccharide (LPS) to investigate the effects of the induced hyperinflammation on the renal tubules. RESULTS: Encapsulated RPTEC/TERT1 cells in Matrigel exhibited elevated renal function markers compared to 2D culture. The coexistence of PBMC and polyI:C induced a strong inflammatory response in the kidney cells. This hyperinflammation significantly reduced primary cilia formation and upregulated kidney injury markers along the 3D tubules. Similarly, treating co-cultured PBMC with LPS to induce hyperinflammation resulted in comparable inflammatory responses and potential kidney injury. CONCLUSION: The model demonstrated similar changes in kidney injury markers following polyI:C and LPS treatment, indicating its suitability for detecting immune-associated kidney damage resulting from infections and biopharmaceutical applications.


Subject(s)
Leukocytes, Mononuclear , Lipopolysaccharides , Humans , Coculture Techniques , Cell Line , Inflammation
3.
Cell Rep ; 30(5): 1447-1462.e5, 2020 02 04.
Article in English | MEDLINE | ID: mdl-32023461

ABSTRACT

Primary cilium is an antenna-like microtubule-based cellular sensing structure. Abnormal regulation of the dynamic assembly and disassembly cycle of primary cilia is closely related to ciliopathy and cancer. The Wnt signaling pathway plays a major role in embryonic development and tissue homeostasis, and defects in Wnt signaling are associated with a variety of human diseases, including cancer. In this study, we provide direct evidence of Wnt3a-induced primary ciliogenesis, which includes a continuous pathway showing that the stimulation of Wnt3a, a canonical Wnt ligand, promotes the generation of ß-catenin p-S47 epitope by CK1δ, and these events lead to the reorganization of centriolar satellites resulting in primary ciliogenesis. We have also confirmed the application of our findings in MCF-7/ADR cells, a multidrug-resistant tumor cell model. Thus, our data provide a Wnt3a-induced primary ciliogenesis pathway and may provide a clue on how to overcome multidrug resistance in cancer treatment.


Subject(s)
Centrioles/metabolism , Cilia/metabolism , Organogenesis , Wnt3A Protein/metabolism , beta Catenin/metabolism , Amino Acid Sequence , Animals , Casein Kinases/metabolism , Centrosome/metabolism , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Epitopes/metabolism , HEK293 Cells , HeLa Cells , Humans , Ligands , MCF-7 Cells , Mice , Phosphorylation , Phosphoserine/metabolism , Wnt3A Protein/chemistry
4.
Yeast ; 30(6): 219-27, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23609041

ABSTRACT

In the post-genomic era, an immediate challenge is to assign biological functions to novel proteins encoded by the genome. This challenge requires the use of a simple organism as a genetic tool and a range of new high-throughput techniques. Schizosacchromyces pombe is a powerful model organism used to investigate disease-related genes and provides useful tools for the functional analysis of heterologous genes. To expand the current array of experimental tools, we constructed two series of Sz. pombe expression vectors, i.e. general and Gateway vectors, containing nourseothricin-resistance markers. Vectors carrying nourseothricin-resistance markers possess advantages in that they do not limit the parental strains with auxotrophic mutations with respect to availability for use in clone selection and can be used together with vectors carrying nutrient markers in minimal media. We modified the pSLF173, pSLF273 and pSLF373 vectors carrying a triple haemagglutinin epitope (3×HA) and an Ura4 marker. The vectors described here contain the nmt1 promoter with three different episomal expression strengths for proteins fused with 3×HA, EGFP or DsRed at the N-terminus. These vectors represent an important contribution to the genome-wide investigation of multiple heterologous genes and for functional and genetic analysis of novel human genes.


Subject(s)
Acetyltransferases/genetics , Drug Resistance, Fungal/genetics , Genetic Vectors/genetics , Schizosaccharomyces/genetics , Streptothricins/pharmacology , Culture Media , Gene Expression Regulation, Fungal , Plasmids/genetics , Promoter Regions, Genetic , Recombinant Fusion Proteins , Schizosaccharomyces/drug effects , Schizosaccharomyces/growth & development
5.
Apoptosis ; 18(7): 870-81, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23546866

ABSTRACT

Shikonin derivatives exert powerful cytotoxic effects, induce apoptosis and escape multidrug resistance in cancer. However, the diverse mechanisms underlying their anticancer activities are not completely understood. Here, we demonstrated that shikonin-induced apoptosis is caused by reactive oxygen species (ROS)-mediated activation of Akt/ASK1/p38 mitogen-activated protein kinase (MAPK) and downregulation of p21(Cip1). In the presence of shikonin, inactivation of Akt caused apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation at Ser83, which is associated with ASK1 activation. Shikonin-induced apoptosis was enhanced by inhibition of Akt, whereas overexpression of constitutively active Akt prevented apoptosis through modulating ASK1 phosphorylation. Silencing ASK1 and MKK3/6 by siRNA reduced the activation of MAPK kinases (MKK) 3/6 and p38 MAPK, and apoptosis, respectively. Antioxidant N-acetyl cysteine attenuated ASK1 dephosphorylation and p38 MAPK activation, indicating that shikonin-induced ROS is involved in the activation of Akt/ASK1/p38 pathway. Expression of p21(Cip1) was significantly induced in early response, but gradually decreased by prolonged exposure to shikonin. Overexpression of p21(Cip1) have kept cells longer in G1 phase and attenuated shikonin-induced apoptosis. Depletion of p21(Cip1) facilitated shikonin-induced apoptosis, implying that p21(Cip1) delayed shikonin-induced apoptosis via G1 arrest. Immunohistochemistry and in vitro binding assays showed transiently altered localization of p21(Cip1) to the cytoplasm by shikonin, which was blocked by Akt inhibition. The cytoplasmic p21(Cip1) actually binds to and inhibits the activity of ASK1, regulating the cell cycle progression at G1. These findings suggest that shikonin-induced ROS activated ASK1 by decreasing Ser83 phosphorylation and by dissociation of the negative regulator p21(Cip1), leading to p38 MAPK activation, and finally, promoting apoptosis.


Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/genetics , Drugs, Chinese Herbal/pharmacology , Gene Expression Regulation, Neoplastic , MAP Kinase Kinase Kinase 5/genetics , Naphthoquinones/pharmacology , Proto-Oncogene Proteins c-akt/genetics , p38 Mitogen-Activated Protein Kinases/genetics , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Humans , MAP Kinase Kinase Kinase 5/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction , p38 Mitogen-Activated Protein Kinases/metabolism
6.
Biochem Biophys Res Commun ; 417(1): 613-8, 2012 Jan 06.
Article in English | MEDLINE | ID: mdl-22182414

ABSTRACT

The small heat-shock protein Hsp9 from Schizosaccharomyces pombe was previously reported to be a homologue of Saccharomyces cerevisiae HSP12. Although Hsp9 is expressed in response to heat shock and nutritional limitation, its function is still not completely understood. Here, we explored the biological function of Hsp9 in S. pombe. The hsp9 gene might play a role in stress adaptation; hsp9 deletion caused heat sensitivity and overexpression induced heat tolerance. In addition, Hsp9 also contribute to cell cycle regulation in the nucleus. Δhsp9 cells grew more quickly and were shorter in length than wild-type cells. Moreover, Δhsp9 cells did not achieve checkpoint arrest under stress conditions, leading to cell death, and exhibited a short doubling time and short G2 phase. Overexpression of hsp9 induced cell cycle delay, increased the population of G2 phase cells, and rescued the phenotypes of cdc2-33, cdc25-22, Δrad24, and Δrad25 mutants, suggesting that Hsp9 probably regulates Cdc2 phosphorylation by modulating the Cdc25 activity. Indeed, immunoprecipitation experiments revealed that Hsp9 is associated with 14-3-3 and Cdc25. In Δhsp9 cells, the association of 14-3-3 with Cdc25 was weakened and Cdc2 phosphorylaton was reduced. Together, our data suggest that Hsp9 has dual functions in stress adaptation and regulating a G2-M checkpoint by the Cdc25 inactivation; this differs from S. cerevisiae HSP12, which maintains cell membrane stability under stress conditions.


Subject(s)
Adaptation, Physiological , G2 Phase Cell Cycle Checkpoints , Heat-Shock Proteins/physiology , Heat-Shock Response , M Phase Cell Cycle Checkpoints , Phosphoprotein Phosphatases/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces pombe Proteins/physiology , 14-3-3 Proteins/metabolism , Heat-Shock Proteins/genetics , Phosphoprotein Phosphatases/genetics , Phosphorylation , Schizosaccharomyces pombe Proteins/genetics
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