10-N Nonyl-Acridine Orange: A fluorescent probe which stains mitochondria independently of their energetic state

Maftah, Abderrahman; Petit, J.; Ratinaud, Marie‐Hélène; Julien, Raymond

doi:10.1016/0006-291x(89)91700-2

Cited by 147 publications

(90 citation statements)

References 23 publications

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“…56 Briefly, cells were incubated at the concentration of 0.5 × 10 6 /ml with 5 μM NAO for 10 min at 37 ºC, washed twice in cold KHB and analyzed using a FACSCalibur flow cytometer.…”

Section: Methodsmentioning

confidence: 99%

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution

et al. 2015

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Tissue homeostasis requires tight regulation of cellular proliferation, differentiation and apoptosis. E2F1 and E2F2 transcription factors share a critical role in tissue homeostasis, since their combined inactivation results in overall organ involution, specially affecting the pancreatic gland, which subsequently triggers diabetes. We have examined the mechanism by which these E2Fs regulate tissue homeostasis. We show that pancreas atrophy in E2F1/E2F2 double-knockout (DKO) mice is associated with mitochondrial apoptosis and activation of the p53 pathway in young animals, before the development of diabetes. A deregulated expression of E2F target genes was detected in pancreatic cells of young DKO animals, along with unscheduled DNA replication and activation of a DNA damage response. Importantly, suppression of DNA replication in vivo with aphidicolin led to a significant inhibition of the p53 pathway in DKO pancreas, implying a causal link between DNA replication stress and p53 activation in this model. We further show that activation of the p53 pathway has a key role in the aberrant phenotype of DKO mice, since targeted inactivation of p53 gene abrogated cellular apoptosis and prevented organ involution and insulin-dependent diabetes in mice lacking E2F1/E2F2. Unexpectedly, p53 inactivation unmasked oncogenic features of E2F1/E2F2-depleted cells, as evidenced by an accelerated tumor development in triple-knockout mice compared with p53 − / − mice. Collectively, our data reveal a role for E2F1 and E2F2 as suppressors of replicative stress in differentiating cells, and uncover the existence of a robust E2F-p53 regulatory axis to enable tissue homeostasis and prevent tumorigenesis. These findings have implications in the design of approaches targeting E2F for cancer therapy.

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“…56 Briefly, cells were incubated at the concentration of 0.5 × 10 6 /ml with 5 μM NAO for 10 min at 37 ºC, washed twice in cold KHB and analyzed using a FACSCalibur flow cytometer.…”

Section: Methodsmentioning

confidence: 99%

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution

et al. 2015

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“…The relative mitochondrial volume was measured using flow cytometry with 10-nnonyl-acridine orange (NAO; Molecular Probes), which has been demonstrated to bind specifically to the mitochondrial inner membrane, independent of its energy state (⌬⌿ m ). 25 For NAO staining, the cells were trypsinized, resuspended in PBS, and fixed in 70% ethanol for 10 minutes at 4°C. After ethanol was removed by centrifugation, the cells were washed in PBS and stained with 10 mol/L NAO in PBS.…”

Section: Methodsmentioning

confidence: 99%

Hydrogen peroxide overproduction in megamitochondria of troglitazone-treated human hepatocytes

Shishido

2003

Hepatology

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Troglitazone has been withdrawn from therapeutic options for diabetes mellitus because of its severe hepatocyte toxicity of unknown pathogenesis. The aim of the present study was to assess both morphologic and functional alterations in the mitochondria of troglitazonetreated hepatocytes. A polarized human hepatocyte cell line, OUMS-29, was used in this study. The mitochondrial volume and the mitochondrial transmembrane potential (⌬⌿ m ) were examined using flow cytometry with nonylacridine orange (NAO) and rhodamine-123, respectively. An ultrastructural examination of the troglitazone-treated OUMS-29 cells was performed using transmission electron microscopy (TEM). Reactive oxygen species (ROS) production was assessed using flow cytometry with dihydroethidium and 2 ,7 -dichlorodihydrofluorescein diacetate. A significant increase in the mitochondrial volume of the troglitazone-treated cells was found by the NAO analysis, in comparison with pioglitazone-treated and ciglitazone-treated cells. The increase in volume was due to a marked enlargement in the mitochondria. The markedly enlarged mitochondria with intramitochondrial electrondense deposits were confirmed on TEM, which showed myelin-like structures, indicating degraded membrane constituents. The troglitazone-treated cells showed a significant decline in the ⌬⌿ m per unit mitochondrial volume but resulted in no clear cell death. ROS analysis revealed a significant production of hydrogen peroxide in the troglitazone-treated hepatocytes. This production was attenuated using an antioxidant, N-acetyl-L-cysteine. In conclusion, troglitazone caused overproduction of hydrogen peroxide, which deteriorated both mitochondrial membrane structures and mitochondrial function, leading to a possible priming for the severe hepatocyte toxicity. (HEPATOLOGY 2003;37:136-147.) T roglitazone, a ligand for peroxisome proliferatoractivated receptor ␥ (PPAR␥), used to be administered as an antidiabetic drug until it was discovered to be associated with hepatotoxicity and withdrawn from the market after reports of several dozens of deaths or cases of severe hepatic failure requiring liver transplantation. 1,2 In contrast to troglitazone, 2 other glitazones, pioglitazone and rosiglitazone, are still on the market; however, several cautionary reports have suggested that the latter 2 drugs show hepatotoxicity as a class effect of glitazones. [2][3][4][5][6][7][8][9] Although the troglitazone-associated hepatotoxicity has been considered idiosyncratic direct hepatocyte toxicity, 10 the precise mechanism has remained unclear. In our recent study, mitochondrial damage was ultrastructurally demonstrated in troglitazone-treated human hepatoma cells, 11 allowing us to first focus on alterations in the mitochondrial structure and the function in the troglitazone-treated human hepatocytes. Troglitazone is known

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“…Binding of NAO is influenced only marginally by mitochondrial transmembrane electric potential and is determined by the availability of binding sites (27). Following the addition of MTT to cell cultures, there was a rapid decrease in mitochondrial NAO fluorescence (Fig.…”

Section: Mtt Reduction and Nao Mitochondrial Bindingmentioning

confidence: 97%

Mitochondrial and nonmitochondrial reduction of MTT: Interaction of MTT with TMRE, JC‐1, and NAO mitochondrial fluorescent probes

2002

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Background: Bioreduction of water-soluble tetrazolium salts (e.g., MTS, XTT, and MTT) to their respective formazans is generally regarded as an indicator of cell "redox activity." The reaction is attributed mainly to mitochondrial enzymes and electron carriers. However, MTT reduction may also be catalyzed by a number of other nonmitochondrial enzymes. The goal of this work was to establish the sites of MTT reduction in intact HepG2 human hepatoma cells in culture. Methods: In order to establish the subcellular localization of the sites of reduction of MTT, we imaged the formation of MTT-formazan deposits using backscattered light confocal microscopy. Mitochondria were visualized in viable cells using fluorescent dyes that bind in a manner dependent (JC-1 and TMRE) or independent (NAO) of mitochondrial electric potential. Results: Only 25-45% of MTT-formazan was associated with mitochondria after 25 min of incubation. No more than 25% of the mitochondrial area on images was occu-

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10-N Nonyl-Acridine Orange: A fluorescent probe which stains mitochondria independently of their energetic state

Cited by 147 publications

References 23 publications

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution

Hydrogen peroxide overproduction in megamitochondria of troglitazone-treated human hepatocytes

Mitochondrial and nonmitochondrial reduction of MTT: Interaction of MTT with TMRE, JC‐1, and NAO mitochondrial fluorescent probes

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