Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry

Troiano, Leonarda; Ferraresi, Roberta; Lugli, Enrico; Nemes, Elisa; Roat, Erika; Nasi, Milena; Pinti, Marcello; Cossarizza, Andrea

doi:10.1038/nprot.2007.405

Cited by 147 publications

(109 citation statements)

References 45 publications

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“…Hoechst 33342 staining was performed as described earlier (19), and cells were analyzed using the CyFlow ML (Partec GmbH, Muenster, Germany) cytofluorimeter. Cells were stained with TO-PRO3 vital dye (Molecular Probes) before analysis as described previously (60). Immunofluorescence was carried out as described by Kilstrup-Nielsen et al (30), and stained cells were observed with a Zeiss AxioSkop 40 fluorescence microscope (Carl Zeiss, Germany).…”

Section: Methodsmentioning

confidence: 99%

HOXD13 Binds DNA Replication Origins To Promote Origin Licensing and Is Inhibited by Geminin

Salsi¹,

Ferrari²,

Ferraresi

et al. 2009

Molecular and Cellular Biology

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HOX DNA-binding proteins control patterning during development by regulating processes such as cell aggregation and proliferation. Recently, a possible involvement of HOX proteins in replication origin activity was suggested by results showing that a number of HOX proteins interact with the DNA replication licensing regulator geminin and bind a characterized human origin of replication. The functional significance of these observations, however, remained unclear. We show that HOXD13, HOXD11, and HOXA13 bind in vivo all characterized human replication origins tested. We furthermore show that HOXD13 interacts with the CDC6 loading factor, promotes pre-replication complex (pre-RC) proteins assembly at origins, and stimulates DNA synthesis in an in vivo replication assay. HOXD13 expression in cultured cells accelerates DNA synthesis initiation in correlation with the earlier pre-RC recruitment onto origins during G 1 phase. Geminin, which interacts with HOXD13 as well, blocks HOXD13-mediated assembly of pre-RC proteins and inhibits HOXD13-induced DNA replication. Our results uncover a function for Hox proteins in the regulation of replication origin activity and reveal an unforeseen role for the inhibition of HOX protein activity by geminin in the context of replication origin licensing.Hox proteins belong to the large family of homeodomaincontaining DNA-binding proteins. During embryonic development they control cell fates, eventually leading to the generation of different regional identities along the primary body and limb axes (17,35). Genetic analyses, including loss-and gainof-function experiments, showed that vertebrate Hox genes modulate morphogenetic processes by controlling crucial aspects such as cell proliferation and aggregation (16). This is particularly true of 5Ј HoxA and HoxD genes, which are involved in limb patterning (reviewed in references 49 and 65). The inactivation of the Hoxd13 gene in mice, for instance, causes a phenotype resulting from defects in the proliferation and/or condensation of limb mesenchymal cells (11,14).Hox proteins have been shown to act as transcription factors, which are thought to regulate sets of target genes by binding to specific DNA sequences within the transcriptional regulatory regions of these (7,33,38,43,50,51,61,62). Recent findings, however, have hinted at a possible additional function for this family of DNA-binding proteins. Hox proteins have been found to associate with the DNA replication licensing regulator geminin (39), and a number of them have been shown to bind the human LaminB2 and other origins of replication, suggesting their possible role in origin definition and/or assembly of the replication machinery (9,13,20). The functional significance of these observations, however, remained elusive.Origins of replication are poorly defined in metazoa. A consensus sequence appears not to be required for replication initiation in human cells, and only a few origins where replication initiates from a localized site in each cell cycle have been well characterize...

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Section: Methodsmentioning

confidence: 99%

HOXD13 Binds DNA Replication Origins To Promote Origin Licensing and Is Inhibited by Geminin

Salsi¹,

Ferrari²,

Ferraresi

et al. 2009

Molecular and Cellular Biology

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“…To study mitochondrial function before cell death, assays evaluating mitochondria were performed after 6 hours of 125 mmol/L H 2 O 2 treatment. The JC-1 assay is a well accepted method of studying MMP (31).…”

Section: Autophagic Protein-depleted Mscs Show Increased Susceptibilimentioning

confidence: 99%

Mesenchymal Stromal Cells Deficient in Autophagy Proteins Are Susceptible to Oxidative Injury and Mitochondrial Dysfunction

Ghanta

Tsoyi

Liu

et al. 2017

Am J Respir Cell Mol Biol

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Oxidative stress resulting from inflammatory responses that occur during acute lung injury and sepsis can initiate changes in mitochondrial function. Autophagy regulates cellular processes in the setting of acute lung injury, sepsis, and oxidative stress by modulating the immune response and facilitating turnover of damaged cellular components. We have shown that mesenchymal stromal cells (MSCs) improve survival in murine models of sepsis by also regulating the immune response. However, the effect of autophagy on MSCs and MSC mitochondrial function during oxidative stress is unknown. This study investigated the effect of depletion of autophagic protein microtubule-associated protein 1 light chain 3B (LC3B) and beclin 1 (BECN1) on the response of MSCs to oxidative stress. MSCs were isolated from wild-type (WT) and LC3B 2/2 or Becn1 1/2 mice. MSCs from the LC3B 2/2 and Becn1 1/2 animals had increased susceptibility to oxidative stress-induced cell death as compared with WT MSCs. The MSCs depleted of autophagic proteins also had impaired mitochondrial function (decreased intracellular ATP, reduced mitochondrial membrane potential, and increased mitochondrial reactive oxygen species production) under oxidative stress as compared with WT MSCs. In WT MSCs, carbon monoxide (CO) preconditioning enhanced autophagy and mitophagy, and rescued the cells from oxidative stress-induced death. CO preconditioning was not able to rescue the decreased survival of MSCs from the LC3B 2/2 and Becn1 1/2 animals, further supporting the tenet that CO exerts its cytoprotective effects via the autophagy pathway.

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“…On the other hand, in apoptotic or unhealthy cells with low MMP, JC-1 remains in the monomeric form, which shows only green fluorescence. The ratio of Red/Green represents the amount of healthy cells [27] .…”

Section: Wwwchinapharcom Wu LX Et Almentioning

confidence: 99%

Curcumin derivative C817 inhibits proliferation of imatinib-resistant chronic myeloid leukemia cells with wild-type or mutant Bcr-Abl in vitro

Wāng

Chen

et al. 2014

Acta Pharmacol Sin

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Aim: To find new kinase inhibitors that overcome the imatinib resistance in treatment of chronic myeloid leukemia (CML), we synthesized C817, a novel derivative of curcumin, and tested its activities against wild-type (WT) and imatinib-resistant mutant Abl kinases, as well as in imatinib-sensitive and resistant CML cells in vitro. Methods: 32D cells harboring WT or mutant Abl kinases (nucleotide binding P-loop mutants Q252H, Y253F, and imatinib contact residue mutant T315I), as well as K562/G01 cells (with whole Bcr-Abl gene amplication) were tested. Kinase activity was measured using Kinase-Glo Luminescent Kinase Assay Platform in recombinant WT and mutant (Q252H, Y253F, and T315I) Abl kinases. Cell proliferation and apoptosis were examined using MTT assay and flow cytometry, respectively. The phosphorylation levels of Bcr-Abl initiated signaling proteins were analyzed using Western blotting. Colony forming units (CFU) growth and long term culture-initiating cells (LTC-ICs) were used to test the effects of C817 on human leukemia progenitor/stem cells. Results: C817 potently inhibited both WT and mutant (Q252H, Y253F, and T315I) Abl kinase activities in a non-ATP competitive manner with the values of IC 50 at low nanomole levels. In consistent with above results, C817 suppressed the growth of both imatinib-sensitive and resistant CML cells, including wild-type K562, K562/G01, 32D-T315I, 32D-Q252H, and 32D-Y253F cells with the values of IC 50 at low micromole levels. C817 (0.5 or 1 μmol/L) dose-dependently inhibited the phosphorylation of Bcr-Abl and downstream proteins STAT-5 and CrkL in imatinib-resistant K562/G01 cells. Furthermore, C817 significantly suppressed CFU growth and LTC-ICs, implicating that C817 could eradiate human leukemia progenitor/stem cells. Conclusion: C817 is a promising compound for treatment of CML patients with Bcr-Abl kinase domain mutations that confer imatinib resistance.

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Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry

Cited by 147 publications

References 45 publications

HOXD13 Binds DNA Replication Origins To Promote Origin Licensing and Is Inhibited by Geminin

HOXD13 Binds DNA Replication Origins To Promote Origin Licensing and Is Inhibited by Geminin

Mesenchymal Stromal Cells Deficient in Autophagy Proteins Are Susceptible to Oxidative Injury and Mitochondrial Dysfunction

Curcumin derivative C817 inhibits proliferation of imatinib-resistant chronic myeloid leukemia cells with wild-type or mutant Bcr-Abl in vitro

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