Analysis of the Mitochondrial Membrane Potential Using the Cationic JC-1 Dye as a Sensitive Fluorescent Probe

Sivandzade, Farzane; Bhalerao, Aditya; Cucullo, Luca

doi:10.21769/bioprotoc.3128

Cited by 586 publications

(441 citation statements)

References 9 publications

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“…The JC-1 staining results obtained in our study also showed that THSG might have the ability to stabilize the mitochondrial membrane potential under conditions of H 2 O 2 -induced free radical damage in UB/OC-2 cells (Figure 5e). JC-1 is a cationic dye that enters and accumulates in negatively charged mitochondria and forms a red fluorescent JC-1 dimer that aggregates in healthy cells with a normal membrane potential [33]. Conversely, because an increased mitochondrial membrane permeability results in less negative mitochondria in unhealthy or apoptotic cells, the JC-1 dye does not accumulate to sufficiently high levels to reach the high concentration required for the formation of JC-1 monomer aggregates and continues to exhibit a green fluorescence [33].…”

Section: Discussionmentioning

confidence: 99%

“…JC-1 is a cationic dye that enters and accumulates in negatively charged mitochondria and forms a red fluorescent JC-1 dimer that aggregates in healthy cells with a normal membrane potential [33]. Conversely, because an increased mitochondrial membrane permeability results in less negative mitochondria in unhealthy or apoptotic cells, the JC-1 dye does not accumulate to sufficiently high levels to reach the high concentration required for the formation of JC-1 monomer aggregates and continues to exhibit a green fluorescence [33]. H 2 O 2 rapidly depolarized the mitochondrial membrane potential, which indicated mitochondrial dysfunction.…”

Section: Discussionmentioning

confidence: 99%

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2,3,4′,5-Tetrahydroxystilbene-2-O-β-D-Glucoside (THSG) Activates the Nrf2 Antioxidant Pathway and Attenuates Oxidative Stress-Induced Cell Death in Mouse Cochlear UB/OC-2 Cells

Lin

Luo

et al. 2020

Biomolecules

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Oxidative stress plays a critical role in the pathogenesis of hearing loss, and 2,3,4 ,5-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) exerts antioxidant effects by inhibiting reactive oxygen species (ROS) generation. With the aim of developing new therapeutic strategies for oxidative stress, this study investigated the protective mechanism of THSG in vitro using a normal mouse cochlear cell line (UB/OC-2). The THSG and ascorbic acid have similar free radical scavenger capacities. H 2 O 2 , but not THSG, reduced the UB/OC-2 cell viability. Moreover, H 2 O 2 might induce apoptosis and autophagy by inducing morphological changes, as visualized by microscopy. As evidenced by Western blot analysis and monodansylcadaverine (MDC) staining, THSG might decrease H 2 O 2 -induced autophagy. According to a Western blotting analysis and Annexin V/PI and JC-1 staining, THSG might protect cells from H 2 O 2 -induced apoptosis and stabilize the mitochondrial membrane potential. Furthermore, THSG enhanced the translocation of nucleus factor erythroid 2-related factor 2 (Nrf2) into the nucleus and increased the mRNA and protein expression of antioxidant/detoxifying enzymes under H 2 O 2 -induced oxidative stress conditions. Collectively, our findings demonstrate that THSG, as a scavenging agent, can directly attenuate free radicals and upregulate antioxidant/detoxifying enzymes to protect against oxidative damage and show that THSG protects UB/OC-2 cells from H 2 O 2 -induced autophagy and apoptosis in vitro.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

2,3,4′,5-Tetrahydroxystilbene-2-O-β-D-Glucoside (THSG) Activates the Nrf2 Antioxidant Pathway and Attenuates Oxidative Stress-Induced Cell Death in Mouse Cochlear UB/OC-2 Cells

Lin

Luo

et al. 2020

Biomolecules

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“…Twenty-four hours later, the JC-1 assay was conducted, followed by flow cytometry. The details were described previously (6). JC-1 dye obtained from Thermo Fisher Scientific (Waltham, MA, USA).…”

Section: Apoptosis Assaymentioning

confidence: 99%

Long Non-coding RNA MIR570MG Causes Regorafenib Resistance in Colon Cancer by Repressing miR-145/SMAD3 Signaling

Fang

Wang

et al. 2020

Front. Oncol.

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An increasing number of studies have shown that long non-coding RNA (lncRNA) dysregulation plays a fundamental role in the development of various cancers, including colon cancer. Nonetheless, the mechanisms of lncRNA in regorafenib-resistance remain unclear. Our research revealed the lncRNA MIR570MG increased in regorafenib-resistant colon cancer cells compared to the regorafenib-sensitive cells. Furthermore, MIR570MG sponged miR-145, which declined in regorafenib-resistant colon cancer cell lines. More importantly, overexpression of miR-145 hampered cell proliferation and retrieved colon cancer regorafenib-sensitivity, contrary to the function of MIR570MG. Dual-luciferase reporter assay confirmed that miR-145 bound to 3 ′-UTR of SMAD3, a transcriptional modulator activated by TGFβ, resulting in blockage of TGFβ /SMAD3-mediated cell growth and cycle progression. Besides, ectopic expression of miR-145 inhibitor in the parental cells endowed resistance to regorafenib. Inversely, knockdown of MIR570MG impoverished resistance against regorafenib. Additionally, overexpression of MIR570MG conquered the suppression of tumor growth by miR-146 and rehabilitated the resistance to regorafenib in HCT116R human colon cancer mouse models. In summary, our findings suggested that MIR570MG promoted regorafenib resistance via releasing SMAD3 from miR-145, leading to activation of SMAD3-mediated signaling pathways.

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“…To further evidence ER stress-induced mitochondrial dysfunction and investigate the impact of EUK-134, normalised respiratory flux control ratios were determined using the six parameters of mitochondrial function [21]. A significant decline was found in respiratory control ratio (RCR), phosphorylating respiration, and coupling efficiency under ER stress, which were inhibited by antioxidant treatment (Figure 2H).…”

Section: Mitochondrial Oxygen Consumption and Mitochondrial Unfoldedmentioning

confidence: 99%

Eukarion-134 attenuates endoplasmic reticulum stress-induced mitochondrial dysfunction in human skeletal muscle cells

Thoma

Lyon

Al-Shanti

et al. 2020

Preprint

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Maladaptive endoplasmic reticulum (ER) stress is associated with modified reactive oxygen species (ROS) generation, altered mitochondrial bioenergetics, and oxidative damage; and is postulated as a potential mechanism involved in the underlying muscle weakness experienced by patients with myositis, an acquired autoimmune neuromuscular disease. In this study, we investigate the impact of ROS generation in an in vitro model of ER stress in skeletal muscle, using the ER stress inducer tunicamycin (24 hours) in presence or absence of a superoxide dismutase/catalase mimetic Eukarion (EUK)-134. ER stress activation, ROS generation, mitochondrial function, biogenesis, morphology and dynamics (fusion/fission) were examined. Tunicamycin induced maladaptive ER stress, validated by stimulation of GRP94, GRP78, CHOP, XBP-1, ERDJ4, and GADD34, which were mostly mitigated by EUK-134 at transcriptional level. ER stress triggered mitochondrial unfolded protein response and promoted mitochondrial dysfunction, described by substantial loss of mitochondrial membrane potential, as well as reduction of respiratory control ratio, reserve capacity, phosphorylating respiration, and coupling efficiency, which was ameliorated by EUK-134. ROS-mediated biogenesis and fusion of mitochondria was evident in presence of tunicamycin, which however, had high propensity of fragmentation, accompanied by upregulated mRNA levels of fission-related markers. Increased cellular ROS generation oxidative stress was observed in response to ER stress that was ameliorated in the presence of EUK-134, even though no changes in mitochondrial superoxide were noticeable. These findings suggest that targeting ROS generation using the superoxide dismutase/catalase mimetic EUK-134 can amend aspects of ER stress-induced changes in mitochondrial dynamics and function. Overall, this study suggests that in instances of chronic ER stress, such as in myositis, quenching ROS generation may be a promising therapy for muscle weakness and dysfunction.

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Analysis of the Mitochondrial Membrane Potential Using the Cationic JC-1 Dye as a Sensitive Fluorescent Probe

Cited by 586 publications

References 9 publications

2,3,4′,5-Tetrahydroxystilbene-2-O-β-D-Glucoside (THSG) Activates the Nrf2 Antioxidant Pathway and Attenuates Oxidative Stress-Induced Cell Death in Mouse Cochlear UB/OC-2 Cells

2,3,4′,5-Tetrahydroxystilbene-2-O-β-D-Glucoside (THSG) Activates the Nrf2 Antioxidant Pathway and Attenuates Oxidative Stress-Induced Cell Death in Mouse Cochlear UB/OC-2 Cells

Long Non-coding RNA MIR570MG Causes Regorafenib Resistance in Colon Cancer by Repressing miR-145/SMAD3 Signaling

Eukarion-134 attenuates endoplasmic reticulum stress-induced mitochondrial dysfunction in human skeletal muscle cells

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