scholarly journals Thioflavin T indicates membrane potential in mammalian cells and can affect it in a blue light dependent manner.

2021 ◽  
Author(s):  
Emily Skates ◽  
Hadrien Delattre ◽  
Zoe Schofield ◽  
Munehiro s Asally ◽  
orkun s soyer
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Blue Light ◽  
Mammalian Cells ◽  
Mitochondrial Membrane ◽  
Light Exposure ◽  
Thioflavin T ◽  
Dependent Manner ◽  
Tht Fluorescence ◽  
Low Concentrations

The fluorescent benzothiazole Thioflavin T (ThT) has a high binding affinity to protein aggregates and is used as a marker for the study of this process, most commonly in the context of neurodegenerative disease research and diagnosis. Recently, this same dye was shown to indicate membrane potential in bacteria due to its cationic nature. This finding prompted a question whether ThT fluorescence is linked to the membrane potential in mammalian cells, which would be important for appropriate utilisation of ThT in research and diagnosis. Here, we show that ThT localises into the mitochondria of HeLa cells in a membrane-potential dependent manner. Specifically, ThT colocalised in cells with a well-established mitochondrial membrane-potential indicator Tetramethylrhodamine methyl ester (TMRM) and gave similar temporal responses as TMRM to treatment with a protonophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). Additionally, we found that presence of ThT together with exposure to blue light (λ=405 nm) exposure, but neither factor alone, caused depolarisation of mitochondrial membrane potential. This depolarisation effect was recapitulated by a mathematical model implementing the potential-dependent distribution of ThT and its light-dependent binding in mitochondria. These results show that ThT can act as a membrane potential dye in mammalian cells, when used at low concentrations and with low blue-light exposure, while it causes dissipation of the mitochondrial membrane potential at higher concentrations and in the presence of blue light excitation. This conclusion motivates a re-evaluation of ThT use at micromolar range in live-cell analyses, while indicating that this dye can enable future studies on the potential connections between membrane potential dynamics and protein aggregation.

scholarly journals The Effect of A2E on the Uptake and Release of Calcium in the Lysosomes and Mitochondria of Human RPE Cells Exposed to Blue Light

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Mao-Mei Luo ◽  
Lin Chen ◽  
Shu Wang ◽  
Chun Zeng ◽  
De-Zhi Li ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Blue Light ◽  
Fluorescence Intensity ◽  
Mitochondrial Membrane ◽  
Light Exposure ◽  
Final Concentration ◽  
Rpe Cells ◽  
Uptake And Release ◽  
Human Rpe Cells

We aimed to explore the effect of N-retinylidene-N-retinylethanolamine (A2E) on the uptake and release of calcium in lysosomes and mitochondria by establishing a model of human retinal pigment epithelial (RPE) cell injury induced by exposure to blue light. Primary human RPE cells were cultured from passages 4 to 6 and exposed to blue light at an intensity of 2000 ± 500 lux for 6 hours. After blue light exposure, the culture was maintained for 24 hours. A2E at a final concentration of 25 μM was added to the culture 2 hours before light exposure, and nifedipine at a final concentration of 10−4 M was added 1 hour before light exposure. The levels of Ca2+ in the cytosol (CaTM/2AM), mitochondria (Rhod/2AM), and lysosomes (LysoTracker Red and Fluo-3/AM) were determined. In order to measure the calcium levels in the different organelles, RPE were imaged using a laser scanning confocal microscope. Moreover, changes in the mitochondrial membrane potential were detected by flow cytometry analysis of JC-1-stained cells. The obtained results revealed that blue light illumination increased the calcium fluorescence intensity in the cytoplasm, mitochondria, and lysosomes of human RPE cells when compared with the control cells ( P < 0.05 ). After A2E treatment, the fluorescence intensity of the calcium in the cytoplasm was further increased ( P < 0.05 ), while that in the mitochondria and lysosomes decreased ( P < 0.05 ). In addition, we observed that nifedipine reduced the fluorescence intensity of calcium in the RPE cells. Our results also showed that the mitochondrial membrane potential in the RPE treated with blue light and A2E was lower than that in the control, blue light, and A2E-treated cells ( P < 0.05 ). Blue light increased calcium levels in the cytoplasm, lysosomes, and mitochondria of RPE cells. A2E damages the lysosomal and mitochondrial membranes, resulting in calcium release into the cytoplasm. Finally, our results demonstrated that both blue light and A2E treatments reduced mitochondrial membrane potential, increasing cytosolic Ca2+ levels, which can contribute to the activation of RPE death.

Synthesis and Biological Evaluation of Novel Heterocyclic Imines Linked Coumarin- Thiazole Hybrids as Anticancer Agents

2019 ◽  
Vol 19 (4) ◽  
pp. 557-566 ◽  
Author(s):  
Nerella S. Goud ◽  
Mahammad S. Ghouse ◽  
Jatoth Vishnu ◽  
Jakkula Pranay ◽  
Ravi Alvala ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Membrane Potential ◽  
Fluorescence Spectroscopy ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Annexin V ◽  
Biological Evaluation ◽  
Dependent Manner ◽  
Dapi Staining ◽  
Dose Dependent

Background: Human Galectin-1, a protein of lectin family showing affinity towards β-galactosides has emerged as a critical regulator of tumor progression and metastasis, by modulating diverse biological events including homotypic cell aggregation, migration, apoptosis, angiogenesis and immune escape. Therefore, galectin-1 inhibitors might represent novel therapeutic agents for cancer. Methods: A new series of heterocyclic imines linked coumarin-thiazole hybrids (6a-6r) was synthesized and evaluated for its cytotoxic potential against a panel of six human cancer cell lines namely, lung (A549), prostate (DU-145), breast (MCF-7 & MDA-MB-231), colon (HCT-15 & HT-29) using MTT assay. Characteristic apoptotic assays like DAPI staining, cell cycle, annexin V and Mitochondrial membrane potential studies were performed for the most active compound. Furthermore, Gal-1 inhibition was confirmed by ELISA and fluorescence spectroscopy. Results: Among all, compound 6g 3-(2-(2-(pyridin-2-ylmethylene) hydrazineyl) thiazol-4-yl)-2H-chromen-2- one exhibited promising growth inhibition against HCT-15 colorectal cancer cells with an IC50 value of 1.28 ± 0.14 µM. The characteristic apoptotic morphological features like chromatin condensation, membrane blebbing and apoptotic body formation were clearly observed with compound 6g on HCT-15 cells using DAPI staining studies. Further, annexin V-FITC/PI assay confirmed effective early apoptosis induction by treatment with compound 6g. Loss of mitochondrial membrane potential and enhanced ROS generation were confirmed with JC-1 and DCFDA staining method, respectively by treatment with compound 6g, suggesting a possible mechanism for inducing apoptosis. Moreover, flow cytometric analysis revealed that compound 6g blocked G0/G1 phase of the cell cycle in a dose-dependent manner. Compound 6g effectively reduced the levels of Gal-1 protein in a dose-dependent manner. The binding constant (Ka) of 6g with Gal-1 was calculated from the intercept value which was observed as 1.9 x 107 M-1 by Fluorescence spectroscopy. Molecular docking studies showed strong interactions of compound 6g with Gal-1 protein. Conclusion: Our studies demonstrate the anticancer potential and Gal-1 inhibition of heterocyclic imines linked coumarin-thiazole hybrids.

Grape Seed Proanthocyanidins Protect N2a Cells against Ischemic Injury via Endoplasmic Reticulum Stress and Mitochondrial-associated Pathways

2019 ◽  
Vol 18 (4) ◽  
pp. 334-341 ◽  
Author(s):  
Kun Fu ◽  
Liqiang Chen ◽  
Lifeng Miao ◽  
Yan Guo ◽  
Wei Zhang ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Cell Viability ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Grape Seed ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Grape Seed Proanthocyanidins ◽  
N2a Cells ◽  
Caspase 12

Background/Objective: Grape seed proanthocyanidins (GSPs) are a group of polyphenolic bioflavonoids, which possess a variety of biological functions and pharmacological properties. We studied the neuroprotective effects of GSP against oxygen-glucose deprivation/reoxygenation (OGD/R) injury and the potential mechanisms in mouse neuroblastoma N2a cells. Methods: OGD/R was conducted in N2a cells. Cell viability was evaluated by CCK-8 and LDH release assay. Apoptosis was assessed by TUNEL staining and flow cytometry. Protein levels of cleaved caspase-3, Bax and Bcl-2 were detected by Western blotting. CHOP, GRP78 and caspase-12 mRNA levels were assessed by real-time PCR. JC-1 dying was used to detect mitochondrial membrane potential. ROS levels, activities of endogenous antioxidant enzymes and ATP production were examined to evaluate mitochondrial function. Results: GSP increased cell viability after OGD/R injury in a dose-dependent manner. Furthermore, GSP inhibited cell apoptosis, reduced the mRNA levels of CHOP, GRP78 and caspase-12 (ER stressassociated genes), restored mitochondrial membrane potential and ATP generation, improved activities of endogenous anti-oxidant ability (T-AOC, GXH-Px, and SOD), and decreased ROS level. Conclusion: Our findings suggest that GSP can protect N2a cells from OGD/R insult. The mechanism of anti-apoptotic effects of GSP may involve attenuating ER stress and mitochondrial dysfunction.

scholarly journals Review of T-2307, an Investigational Agent That Causes Collapse of Fungal Mitochondrial Membrane Potential

2021 ◽  
Vol 7 (2) ◽  
pp. 130
Author(s):  
Nathan P. Wiederhold
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Candida Species ◽  
Mammalian Cells ◽  
Mitochondrial Membrane ◽  
Fungal Infections ◽  
Published Data ◽  
Candida Auris

Invasive infections caused by Candida that are resistant to clinically available antifungals are of increasing concern. Increasing rates of fluconazole resistance in non-albicans Candida species have been documented in multiple countries on several continents. This situation has been further exacerbated over the last several years by Candida auris, as isolates of this emerging pathogen that are often resistant to multiple antifungals. T-2307 is an aromatic diamidine currently in development for the treatment of invasive fungal infections. This agent has been shown to selectively cause the collapse of the mitochondrial membrane potential in yeasts when compared to mammalian cells. In vitro activity has been demonstrated against Candida species, including C. albicans, C. glabrata, and C. auris strains, which are resistant to azole and echinocandin antifungals. Activity has also been reported against Cryptococcus species, and this has translated into in vivo efficacy in experimental models of invasive candidiasis and cryptococcosis. However, little is known regarding the clinical efficacy and safety of this agent, as published data from studies involving humans are not currently available.

scholarly journals Mechanism of apoptosis induced by quinoxalone from the myxobacterium Stigmatella eracta WXNXJ-B in B16 mouse melanoma cell line

2017 ◽  
Author(s):  
Dahong Wang ◽  
Lanlan Wei ◽  
Shuaiying Zhang
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Biological Activities ◽  
Chromatin Condensation ◽  
Fluorescent Microscopy ◽  
Dependent Manner ◽  
Dna Ladder ◽  
Mouse Melanoma ◽  
B16 Mouse Melanoma

The biological activities of quinoxalone, a novel small molecular substance isolated from the broth of the myxobacterium Stigmatella eracta WXNXJ-B, was investigated. This study was designed to determine the anti-proliferative, apoptotic property of quinoxalone, using B16 mouse melanoma cells as a model system. The results showed that quinoxalone has antitumor activity and can significantly inhibit the proliferation of B16 cells. The extent and the timing of apoptosis were strongly dependent on the dose. After treating with quinoxalone and staining with Hoechst 33342, B16 cells showed typical apoptotic morphological features such as chromatin condensation by fluorescent microscopy. DNA isolated from B16 cells cultured with quinoxalone showed a typical DNA ladder of apoptosis in agarose gel electrophoresis. Further investigation results showed that the apoptotic machinery of B16 induced by quinoxalone was associated with drop in mitochondrial membrane potential from 5.35% to 23.7%, up-regulation of Bax and down-regulation of Bcl-2 in a dose-dependent manner. And a significant increased activation of caspase-3. Our finding suggests that quinoxalone could suppress the growth of B16 cells and reduces cell survival via disturbing mitochondrial membrane potential and inducing apoptosis of tumor cells.

scholarly journals K+/H+-antiporter nigericin arrests DNA synthesis in Ehrlich ascites carcinoma cells

1989 ◽  
Vol 86 (17) ◽  
pp. 6626-6629 ◽  
Author(s):  
L B Margolis ◽  
Y u Novikova I ◽  
I A Rozovskaya ◽  
V P Skulachev
Keyword(s):  
Membrane Potential ◽  
Dna Synthesis ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Low Concentrations ◽  
Ehrlich Ascites ◽  
Inhibition Of Dna Synthesis ◽  
Dna Synthesis Inhibition

Acidification of the cytoplasm of Ehrlich ascites carcinoma cells to pH 6.3 arrests DNA synthesis in these cells. Such an effect can be achieved by incubating the cells at pH 6.2 or by adding low concentrations of the K+/H+ antiporter, the antibiotic nigericin, at neutral pH. Glucose and anaerobiosis potentiate the nigericin effect. The inhibition of DNA synthesis by nigericin occurs without any significant decrease in the ATP concentration and in the mitochondrial membrane potential. The DNA synthesis inhibition is caused neither by a decrease in the intracellular [K+] nor by an increase in the intracellular [Na+] accompanying the nigericin effect (at least at low concentrations of the antibiotic). Nigericin should thus be regarded as a type of a cytostatic primarily affecting intracellular pH.

Metformin Collaborates With PINK1/Mfn2 Overexpression Prevented Isoproterenol-Induced Cardiomyocyte Injury By Improving Mitochondrial Function

2021 ◽  
Author(s):  
Zhuang Ma ◽  
Zuheng Liu ◽  
Yuting Xue ◽  
Hao Zhang ◽  
Wenjun Xiong ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Energy Metabolism ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Biogenesis ◽  
Mitochondrial Membrane ◽  
Mitochondrial Morphology ◽  
Dependent Manner ◽  
Combination Strategy ◽  
Mitochondrial Energy Metabolism ◽  
Atp Production

Abstract Background: Both mitochondrial quality control and energy metabolism are critical in maintaining the physiological function of cardiomyocytes. Previous studies indicated that PGC-1α is a transcription co-activator in promoting mitochondrial energy metabolism which would be beneficial for cardiomyocytes. However, PGC-1α overexpression in heart tissues could also result in the development of cardiomyopathy. This discrepancy in vivo and in vitro might be due to neglecting the elimination of damaged mitochondrial. Thus, an integration strategy of mitochondrial biogenesis and mitophagy might be beneficial.Methods: We studied the function of PINK1 in mitophagy in isoproterenol (Iso)-induced cardiomyocyte injury. Adenovirus was used to provoke an overexpression of the PINK1/Mfn2 protein. Mitochondrial morphology was examined via electron microscopy and confocal microscopy. Cardiomyocytes injury were measured by mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and apoptosis. Metformin was used to increase mitochondrial biogenesis, the level of which was detected via immunoblotting. Additionally, mitochondrial respiratory function was measured by ATP production and oxygen consumption rate (OCR). Results: Cardiomyocytes treated with Iso had high levels of PINK1 and low levels of Mfn2 in a time-dependent manner. PINK1 overexpression promoted mitophagy, alleviated Iso-induced reduction in MMP, reduced ROS production and the apoptotic rate. In addition to increasing mitophagy, metformin could promote mitochondrial biogenensis and the overexpression of Mfn2 induce mitochondrial fusion. Moreover, metformin treatment and PINK1/Mfn2 overexpression reduced the mitochondrial dysfunction by inhibiting the generation of ROS, and leading to an increase in both ATP production and mitochondrial membrane potential in Iso-induced cardiomyocytes injury. Conclusion: Our findings indicate that a combination strategy may help ameliorate myocardial injury through mitophagy and mitochondrial biogenesis.

Encircling granulosa cells protects against di-(2-ethylhexyl)phthalate-induced apoptosis in rat oocytes cultured in vitro

Zygote ◽  
2019 ◽  
Vol 27 (4) ◽  
pp. 203-213 ◽  
Author(s):  
Anima Tripathi ◽  
Vivek Pandey ◽  
A.N. Sahu ◽  
Alok K. Singh ◽  
Pawan K. Dubey
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Granulosa Cells ◽  
Mitochondrial Membrane ◽  
Dependent Manner ◽  
Apoptotic Markers ◽  
Induced Apoptosis ◽  
Ethylhexyl Phthalate

SummaryThe present study investigated if the presence of encircling granulosa cells protected against di(2-ethylhexyl)phthalate (DEHP)-induced oxidative stress in rat oocytes cultured in vitro. Denuded oocytes and cumulus–oocyte complexes (COCs) were treated with or without various doses of DEHP (0.0, 25.0, 50.0, 100, 200, 400 and 800 μM) in vitro. Morphological apoptotic changes, levels of oxidative stress and reactive oxygen species (ROS), mitochondrial membrane potential, and expression levels of apoptotic markers (Bcl2, Bax, cytochrome c) were analyzed. Our results showed that DEHP induced morphological apoptotic changes in a dose-dependent manner in denuded oocytes cultured in vitro. The effective dose of DEHP (400 µg) significantly (P>0.05) increased oxidative stress by elevating ROS levels and the mitochondrial membrane potential with higher mRNA expression and protein levels of apoptotic markers (Bax, cytochrome c). Encircling granulosa cells protected oocytes from DEHP-induced morphological changes, increased oxidative stress and ROS levels, as well as increased expression of apoptotic markers. Taken together our data suggested that encircling granulosa cells protected oocytes against DEHP-induced apoptosis and that the presence of granulosa cells could act positively towards the survival of oocytes under in vitro culture conditions and may be helpful during assisted reproductive technique programmes.

Visualization of mitochondrial membrane potential in mammalian cells

2020 ◽  
pp. 221-245
Author(s):  
Noemí Esteras ◽  
Merel J.W. Adjobo-Hermans ◽  
Andrey Y. Abramov ◽  
Werner J.H. Koopman
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mammalian Cells ◽  
Mitochondrial Membrane
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