Acridine Orange/Ethidium Bromide (AO/EB) Staining to Detect Apoptosis

Kasibhatla, Shailaja; Amarante-Mendes, Gustavo P.; Finucane, Deborah; Brunner, Thomas; Bossy‐Wetzel, Ella; Green, Douglas R.

doi:10.1101/pdb.prot4493

Cited by 376 publications

(205 citation statements)

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“…8A). Cells are classified into four different groups, i.e., preapoptotic, late apoptotic, necrotic, and live cells (49). The results clearly demonstrated that AK-B-transfected cells were more prone to apoptotic induction than cells with EBNA3C either alone or in combination with AK-B, as seen by a 2-to 4-fold drop in apoptotic levels (Fig.…”

Section: Mdm2mentioning

confidence: 62%

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EBNA3C-Mediated Regulation of Aurora Kinase B Contributes to Epstein-Barr Virus-Induced B-Cell Proliferation through Modulation of the Activities of the Retinoblastoma Protein and Apoptotic Caspases

Jha

Saha

et al. 2013

J Virol

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Epstein-Barr virus (EBV) is an oncogenic gammaherpesvirus that is implicated in several human malignancies, includingBurkitt's lymphoma (BL), posttransplant lymphoproliferative disease (PTLD), nasopharyngeal carcinoma (NPC), and AIDSassociated lymphomas. Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential EBV latent antigens, can induce mammalian cell cycle progression through its interaction with cell cycle regulators. Aurora kinase B (AK-B) is important for cell division, and deregulation of AK-B is associated with aneuploidy, incomplete mitotic exit, and cell death. Our present study shows that EBNA3C contributes to upregulation of AK-B transcript levels by enhancing the activity of its promoter. Further, EBNA3C also increased the stability of the AK-B protein, and the presence of EBNA3C leads to reduced ubiquitination of AK-B. Importantly, EBNA3C in association with wild-type AK-B but not with its kinase-dead mutant led to enhanced cell proliferation, and AK-B knockdown can induce nuclear blebbing and cell death. This phenomenon was rescued in the presence of EBNA3C. Knockdown of AK-B resulted in activation of caspase 3 and caspase 9, along with poly(ADP-ribose) polymerase 1 (PARP1) cleavage, which is known to be an important contributor to apoptotic signaling. Importantly, EBNA3C failed to stabilize the kinasedead mutant of AK-B compared to wild-type AK-B, which suggests a role for the kinase domain in AK-B stabilization and downstream phosphorylation of the cell cycle regulator retinoblastoma protein (Rb). This study demonstrates the functional relevance of AK-B kinase activity in EBNA3C-regulated B-cell proliferation and apoptosis.

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Section: Mdm2mentioning

confidence: 62%

“…8A and B). To specifically visualize the apoptotic cells and quantitatively distinguished them from necrotic cells, we performed apoptosis assays using a staining strategy with ethidium bromide and acridine orange (49,50) (Fig. 8A).…”

Section: Mdm2mentioning

confidence: 99%

EBNA3C-Mediated Regulation of Aurora Kinase B Contributes to Epstein-Barr Virus-Induced B-Cell Proliferation through Modulation of the Activities of the Retinoblastoma Protein and Apoptotic Caspases

Jha

Saha

et al. 2013

J Virol

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“…The ability of pneumolysin to kill statin-treated cells in vitro was determined using fluorescence microscopy to detect acridine orange (AO) and ethidium bromide (EtBr) staining (44). HBMEC monolayers in 24-well plates at 20%-40% confluence were treated overnight with 1 μg/ml simvastatin, washed, and incubated with serumfree medium containing 0.5 μg/ml pneumolysin, AO (100 mg/ml), and EtBr (100 mg/ml).…”

Section: Methodsmentioning

confidence: 99%

Statins protect against fulminant pneumococcal infection and cytolysin toxicity in a mouse model of sickle cell disease

et al. 2010

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Sickle cell disease (SCD) is characterized by intravascular hemolysis and inflammation coupled to a 400-fold greater incidence of invasive pneumococcal infection resulting in fulminant, lethal pneumococcal sepsis. Mechanistically, invasive infection is facilitated by a proinflammatory state that enhances receptor-mediated endocytosis of pneumococci into epithelial and endothelial cells. As statins reduce chronic inflammation, in addition to their serum cholesterol-lowering effects, we hypothesized that statin therapy might improve the outcome of pneumococcal infection in SCD. In this study, we tested this hypothesis in an experimental SCD mouse model and found that statin therapy prolonged survival following pneumococcal challenge. The protective effect resulted in part from decreased platelet-activating factor receptor expression on endothelia and epithelia, which led to reduced bacterial invasion. An additional protective effect resulted from inhibition of host cell lysis by pneumococcal cholesterol-dependent cytotoxins (CDCs), including pneumolysin. We conclude therefore that statins may be of prophylactic benefit against invasive pneumococcal disease in patients with SCD and, more broadly, in settings of bacterial pathogenesis driven by receptor-mediated endocytosis and the CDC class of toxins produced by Gram-positive invasive bacteria.

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“…Then, cells were treated with different concentrations of PCN-J04 for 24 h. The culture medium was aspirated from each well and cells were gently rinsed twice with PBS and subsequently treated with 100 ll of ethidium bromide and acridine orange (1:1 w/w) (Kasibhatla et al 2006). Viewed Estimation of lipids by H 1 NMR Eighty percentage confluence of cells were incubated in presence of 7.66 lg/mL of final concentration of peptide containing PCN-J04 for 48 h, and the cellular lipids were isolated by previously described method (Folch et al 1957).…”

Section: Apoptosis Analysis Using Duel Stainingmentioning

confidence: 99%

pACC1 peptide loaded chitosan nanoparticles induces apoptosis via reduced fatty acid synthesis in MDA-MB-231 cells

et al. 2015

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The development of formulations with therapeutic peptides has been restricted to poor cell penetration and in this attempt; we developed pACC1 peptide loaded chitosan nanoparticles. The prepared nanoparticles were characterized with FT-IR, XRD, SEM and TEM. In addition, the suitable formulation was evaluated for hemocompatibility, plasma stability and embryo toxicity using Danio rerio embryo model. The results showed that pACC1 peptide loaded chitosan nanoparticles were compatible with plasma. They possess sustained release pattern and also found to be safe up to 300 mg/L in embryo toxicity tests. Cytotoxicity assays with MDA-MB-231 cell lines suggested that, pACC1 peptide loaded chitosan nanoparticles were capable of enhanced cellular penetration and reduced palmitic acid content, which was confirmed by H 1 NMR . Hence, these nanoparticles could be employed as excellent adjuvant therapeutics while treating solid tumors with multi-drug resistance.

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Acridine Orange/Ethidium Bromide (AO/EB) Staining to Detect Apoptosis

Cited by 376 publications

References 0 publications

EBNA3C-Mediated Regulation of Aurora Kinase B Contributes to Epstein-Barr Virus-Induced B-Cell Proliferation through Modulation of the Activities of the Retinoblastoma Protein and Apoptotic Caspases

EBNA3C-Mediated Regulation of Aurora Kinase B Contributes to Epstein-Barr Virus-Induced B-Cell Proliferation through Modulation of the Activities of the Retinoblastoma Protein and Apoptotic Caspases

Statins protect against fulminant pneumococcal infection and cytolysin toxicity in a mouse model of sickle cell disease

pACC1 peptide loaded chitosan nanoparticles induces apoptosis via reduced fatty acid synthesis in MDA-MB-231 cells

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