Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells

Chen, Shun Hua; Chao, Angel; Tsai, Chia-Lung; Sue, Shih‐Che; Lin, Chiao-Yun; Lee, Yi-Zong; Hung, Yi-Lin; Chao, An‐Shine; Cheng, Ann‐Joy; Wang, Hsin-Shih; Wang, Tzu‐Hao

doi:10.1016/j.omtm.2018.12.005

Cited by 13 publications

(15 citation statements)

References 49 publications

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“…Furthermore, we supplemented the medium with HEPES and mannitol, based on a recommendation for transfection with the commercially available CS-based transfection reagent, Novafect, from NovaMatrix ® (Sandvika, N). Interestingly, HEPES has been reported to be beneficial for transfection purposes before [45,46]. As expected, nanocomplexes were relatively stable in Opti-MEM™ with supplements in contrast to nanocomplexes in the pure medium, even though the size of the nanocomplexes increased slightly ( Figure 5).…”

Section: Supplemented Transfection Medium Stabilizes Nanocomplexes Busupporting

confidence: 52%

Chitosan Nanocomplexes for the Delivery of ENaC Antisense Oligonucleotides to Airway Epithelial Cells

et al. 2020

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Nanoscale drug delivery systems exhibit a broad range of applications and promising treatment possibilities for various medical conditions. Nanomedicine is of great interest, particularly for rare diseases still lacking a curative treatment such as cystic fibrosis (CF). CF is defined by a lack of Cl− secretion through the cystic fibrosis transmembrane conductance regulator (CFTR) and an increased Na+ absorption mediated by the epithelial sodium channel (ENaC). The imbalanced ion and water transport leads to pathological changes in many organs, particularly in the lung. We developed a non-viral delivery system based on the natural aminopolysaccharide chitosan (CS) for the transport of antisense oligonucleotides (ASO) against ENaC to specifically address Na+ hyperabsorption. CS–ASO electrostatic self-assembled nanocomplexes were formed at varying positive/negative (P/N) charge ratios and characterized for their physicochemical properties. Most promising nanocomplexes (P/N 90) displayed an average size of ~150 nm and a zeta potential of ~+30 mV. Successful uptake of the nanocomplexes by the human airway epithelial cell line NCI-H441 was confirmed by fluorescence microscopy. Functional Ussing chamber measurements of transfected NCI-H441 cells showed significantly decreased Na+ currents, indicating successful downregulation of ENaC. The results obtained confirm the promising characteristics of CS as a non-viral and non-toxic delivery system and demonstrate the encouraging possibility to target ENaC with ASOs to treat abnormal ion transport in CF.

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Section: Supplemented Transfection Medium Stabilizes Nanocomplexes Busupporting

confidence: 52%

Chitosan Nanocomplexes for the Delivery of ENaC Antisense Oligonucleotides to Airway Epithelial Cells

et al. 2020

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“…Anti-STIP1 antibodies were delivered into cancer cells using the HEPES method ( 25 ) and subsequently stained with Alexa Fluor 488-conjugated secondary antibodies. The intracellular penetration of antibodies induced by HEPES was confirmed in ovarian cancer cells (MDAH2774) using confocal microscopy ( Figure 1A ).…”

Section: Resultsmentioning

confidence: 99%

“…The transfection mixture was added to cultured cells in Opti-MEM without serum and incubated at 37 °C for 4–24 h. Briefly, the proteins and HEPES were mixed and incubated for 15 min, and the transfected proteins were detected using fluorescent microscopy after 24 h. The amount of individual components in the transfection mixtures was adjusted according to the size of the cultures. For example, when using a 6-well plate, 4 µg of protein were mixed with 200 µL of HEPES ( 25 ).…”

Section: Methodsmentioning

confidence: 99%

“…Following incubation for 24 h, the cells were centrifuged at 300 × g for 5 min, the supernatant was discarded, and the cells attached to the plates were analyzed using a commercially available [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay (Promega, Madison, WI, USA). Cell toxicity was assessed using the lactate dehydrogenase (LDH) method (Sigma-Aldrich) ( 25 ). Since the MTT assay is more sensitive than the LDH assay ( 26 , 27 ), the cells subjected to transfection were incubated for 48 h and centrifuged at 300 × g for 5 min.…”

Section: Methodsmentioning

confidence: 99%

“…It has also been demonstrated that intracellular STIP1 mediates the heat-induced spread of hepatocellular carcinoma to distant sites by facilitating epithelial-mesenchymal transition via an increased nuclear shuttling of Snail1 ( 24 ). In addition, the exposure of cells to 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) is an effective means to promote the intracellular delivery of either antibodies or small peptides ( 25 ).…”

Section: Introductionmentioning

confidence: 99%

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Intracellular targeting of STIP1 inhibits human cancer cell line growth

Lin¹,

Chen²,

Tsai³

et al. 2021

Transl Cancer Res TCR

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Background: Extracellular and cell-surface molecules remain the most common druggable cancer targets.However, intracellular therapeutic modalities are gaining momentum. The overexpression of stress-induced phosphoprotein 1 (STIP1), an adaptor protein that coordinates the functions of different chaperones in protein folding, has been reported in several solid malignancies. Here, we investigated the effects of intracellular STIP1 inhibition, attained either through the HEPES-mediated cytosolic delivery of anti-STIP1 antibodies or the use of a cell-penetrating signal-tagged peptide 520, in different human cancer cell lines and luciferase-expressing murine ovarian cancer cells (MOSEC/Luc) tumor-bearing C57BL/6 mice. Methods:The effects of STIP1 in different human cell lines were determined by cell viability, cell cytotoxicity and cell apoptosis assays. Immunoblotting was used to assess the relevant proteins found in this study and tumor xenograft mice models were also employed.Results: Intracellular targeting of STIP1 inhibited cancer cell line growth and promoted caspase 3-dependent apoptotic cell death. Moreover, the intracellular delivery of anti-STIP1 antibodies facilitated the degradation of STIP1 and two of its client proteins, lysine-specific demethylase 1 and Janus kinase 2.In vivo studies demonstrated that survival of mice bearing experimental tumors was improved by administration of anti-STIP1 antibodies.Conclusions: Our findings demonstrate that the cytosolic inhibition of STIP1 in tumor cells is feasible and provides a solid basis for further investigation of STIP1 as an intracellular cancer target. Our findings demonstrate that cytosolic inhibition of STIP1 in tumor cells is feasible and provide a solid basis for further exploration of STIP1 as an intracellular cancer target.

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Endoplasmic reticulum stress associated with lead (Pb)‐induced olfactory epithelium toxicity in an olfactory dark basal cell line

Han,

Kamogashira,

Kikuta

et al. 2023

FEBS Open Bio

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Lead (Pb) can damage organs and also have undesirable effects on neural development. To explore the effects of Pb on olfactory cells, we investigated Pb‐induced cell toxicity in the DBC1.2 olfactory cell line, which a focus on endoplasmic reticulum (ER) stress, apoptosis and necroptosis. Representative markers of ER stress, apoptosis and necroptosis were analyzed by quantitative PCR. The mRNA expression levels of GRP94, GRP78, spliced XBP1, PERK and ATF6 increased significantly after Pb exposure in a dose‐dependent manner. The expression of Caspase 3 and Caspase 12 did not increase after Pb exposure, which suggested that apoptosis‐induced cell death was not activated after Pb exposure. However, the mRNA of RIPK3 and MLKL showed increases in expression, which indicated that necroptosis‐induced cell death was activated after Pb exposure. These results indicate that Pb exposure induced dose‐dependent cytotoxicity through ER stress and necroptosis pathways in DBC1.2 cells, whereas the apoptosis pathway was not significantly stimulated. HEPES buffer showed a partial protective effect in terms of ER stress, apoptosis and necroptosis. In summary, the necroptosis pathway plays a crucial rule in Pb exposure‐induced cytotoxicity in olfactory cells.

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Utilization of HEPES for Enhancing Protein Transfection into Mammalian Cells

Cited by 13 publications

References 49 publications

Chitosan Nanocomplexes for the Delivery of ENaC Antisense Oligonucleotides to Airway Epithelial Cells

Chitosan Nanocomplexes for the Delivery of ENaC Antisense Oligonucleotides to Airway Epithelial Cells

Intracellular targeting of STIP1 inhibits human cancer cell line growth

Endoplasmic reticulum stress associated with lead (Pb)‐induced olfactory epithelium toxicity in an olfactory dark basal cell line

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