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CCCP affects the protein synthesis reactions in seedling mitochondria. CCCP causes an uncoupling of the proton gradient that is established during the normal activity of electron carriers in the electron transport chain. The chemical acts essentially as an ionophore and reduces the ability of ATP synthase to function optimally Immunoprecipitation - CCCP, Mitochondrial oxidative phosphorylation uncoupler (ab141229) PINK1 was immunoprecipitated from 0.35 mg of HeLa (human epithelial cell line from cervix adenocarcinoma) (treated with 10uM carbonyl cyanide 3-chlorophenylhydrazone (CCCP. ab141229) for 24 hours) whole cell lysate with ab216144 at 1/30 dilution The mitochondria in such CCCP-treated cells showed loss of ΔΨ m and morphological swelling . Significantly, mitochondria preloaded in vivo with calcein ( M r 623) released this marker dye upon treatment of cells with CCCP; this release was inhibited by cyclosporin A [28] , thus confirming the response as an authentic MPT [4] in intact cells CCCP induces vasorelaxation by a mechanism that does not involve K ATP channel activation in smooth muscle cells of arteries. Mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone induces vasorelaxation without involving K ATP channel activation in smooth muscle cells of arterie

Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) is a protonophore that renders the mitochondrial inner membrane permeable for protons and causes dissipation of the proton gradient across the inner mitochondrial membrane. CCCP also causes uncoupling of mitochondria, which means that the transfer of electrons through the electron transport chain is no longer coupled to ATP production, due to loss of the electrochemical gradient The mitochondria in such CCCP‐treated cells showed loss of ΔΨ m and morphological swelling . Significantly, mitochondria preloaded in vivo with calcein ( M r 623) released this marker dye upon treatment of cells with CCCP; this release was inhibited by cyclosporin A [ 28 ] , thus confirming the response as an authentic MPT [ 4 ] in intact cells CCCP treatment was found to increase the Mt number in the modified maturation medium in which mitochondrial degradation was inhibited by MG132, whereas CCCP treatment did not affect the Mt number in the maturation medium lacking MG132. The relative gene expression of TFAM was furthermore shown to be significantly higher in CCCP-treated oocytes than in untreated oocytes. Taken together, the finding presented here suggest that when the mitochondria are injured, mitochondrial biogenesis and.

Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) induces mitophagy in mammalian cells. CCCP suppresses the expression of few electron transport chain (ETC) proteins. Protonophore (H + ionophore) and uncoupler of oxidative phosphorylation in mitochondria. Shown to have a number of effects on cellular calcium. Inhibits secretion of hepatic lipase and partially inhibits the pH gradient-activated C cccp induced a significant leakage of calcein from mitochondria to cytosol and nucleus, indicating a change in mitochondrial inner membrane permeability. Cyclosporin A (CsA), a transition pore blocker, did not prevent the cccp-induced MPT or the cccp-evoked apoptotic cell death, suggesting that cccp-induced apoptotic process wa Collapsing of the mitochondrial membrane potential by CCCP reduced MTR fluorescence emission in both control (upper row) and PV15 MDCK (lower row) cells; the decrease was more pronounced in the PV-expressing PV15 clone. View Article: PubMed Central - PubMe

Carbonyl cyanide m-chlorophenyl hydrazone - Wikipedi

  1. After CCCP treatment of OMA1 −/− MEFs, overexpressed OMA1‐Flag was still cleaved due to self‐processing (Fig 1F), while cleavage of OMA1‐E324Q‐Falg was completely inhibited in the presence of CCCP (Fig 1F), demonstrating that OMA1 is autocatalytically cleaved upon mitochondrial membrane depolarization
  2. JC-1 Dye is available as a standalone reagent (Cat. No. T3168) or in the MitoProbe JC-1 Assay Kit which contains the JC-1 Dye, CCCP (a mitochondria membrane potential disrupter in DMSO), 10x PBS, and DMSO. Learn more about the JC-1 Dye for mitochondria membrane potentia
  3. CCCP acts as an ionophore, causes a decrease in proton motive force developed in the mitochondrial inner membrane, and reduces ATP synthesis by anaerobic respiration, in contrast to rotenone, which directly inhibits mitochondrial complex I. Therefore, mitochondrial fatty acid synthesis would not be suppressed even in the presence of CCCP because electron flow through complex I is not inhibited.

At the population level, we observed that the first round of CCCP treatment induced a large increase in mitochondrial EYFP-Parkin, and, although small decreases in mitochondrial EYFP-Parkin were observed during the intervals between CCCP treatments, mitochondrial EYFP-Parkin did not drop back to basal levels throughout the duration of the experiment (Fig. 9c). In this regard, the pulses of mitochondrial PINK1 induced by each round of CCCP treatment served to maintain mitochondrial. As shown in Fig. 1a, b and Supplementary Fig. 1a-b, CCCP-induced mitochondria ubiquitination, reduction of mitochondrial mass as well as TOM20 ubiquitination and degradation were attenuated by SHP2.. CCCP is a potent mitochondrial membrane uncoupler. CCCP treatment significantly decreased oocyte ATP content in both the young and aged groups (Fig 3: young; vehicle vs. CCCP: 1.9 ± 0.16 vs. 0.61 ± 0.08, aged; vehicle vs. CCCP: 2.5 ± 0.17 vs. 0.77 ± .0.08, P < 0.001)

CCCP, Mitochondrial oxidative phosphorylation uncoupler

CCCP-treated mitochondria showed not only a rounded ring shape, but also composite features of both ring and tubular shapes, similar to the features of normal mitochondria (Fig. 1b6 and. Mitochondrial uncoupling, with low concentration FCCP, induces ROS-dependent cardioprotection independent of K ATP channel activation Jonathan P. Brennan, Jonathan P. Brennan a Cardiac Physiology (Cardiovascular Division), The Rayne Institute, St Thomas' Hospital, King's College London, SE1 7EH, UK. Search for other works by this author on: Oxford Academic. PubMed. Google Scholar. Richard. CCCP impairs the interaction between STING and TBK1 and concomitantly triggers mitochondria fission. Importantly, the knockout of the crucial mitochondria fission regulator Drp1 restored the STING activity, indicating that CCCP down-modulates the STING pathway through DRP1-mediated mitochondria fragmentation. The protonophore CCCP that disrupts membrane potential suppresses the DMXAA-triggered. These results suggest that in contrast to FL, the CCCP-stabilized form of PINK1 that localizes to the outer mitochondrial membrane facing the cytosol (Narendra et al., 2010), the 60-kD ΔMTS-PINK1, which appears in the absence of PARL, and the 52-kD PINK1 are protease-protected within polarized mitochondria (Fig. 1 d) CCCP is a protonophore which uncouples oxidative phosphorylation, induces ROS and depolarizes mitochondrial membrane potential, thus, triggering mitophagy and bulk autophagy . Researchers have typically challenged cells with CCCP to initiate the autophagy for assessing the oxidative stress induced autophagic flux and mitophagy. Bafilomycin is commonly used to inhibit autophagy by targeting.

The protonophore CCCP induces mitochondrial permeability

CCCP, an uncoupler and antimycin A, an inhibitor of the mitochondrial electron transport chain, inhibited IL-1β release induced by ATP but not by other stimuli. CCCP did not inhibit the ATP-induced generation of reactive oxygen species and cell death, both of which are known to promote IL-1β release, but did inhibit the ATP-induced activation of caspase-1, a component of the NLRP3. Biological description FCCP is an protonophore which is widely used to investigate the role of mitochondria in cellular function. FCCP is a potent uncoupler of mitochondrial oxidative phosphorylation. FCCP disrupts ATP synthesis by transporting protons across the mitochondrial inner membrane, interfering with the proton gradient Results: CCCP induces mitochondrial fragmentation in C2C12 cells. In response to a mitochondrial uncoupler, endogenous autophagosome formation is significantly increased (P < 0.001). Co-localization analyses of confocal images indicate that fragmented mitochondria undergo engulfment by autophagosomes. Conclusion: The findings of this study will enhance our understanding of the mitophagic. In M17 cells stably transduced with PINK1 shRNA, YFP-Parkin translocated to mitochondria in 4.7±1.2% of CCCP-treated cells, whereas 67.3±3.1% of control shRNA M17 cells displayed mitochondrial YFP-Parkin after treatment with 10 µM CCCP for 3 h (p-value <0.001) (Figure 3D and 3E). Vehicle treatment failed to induce YFP-Parkin translocation to mitochondria in both cell lines. The necessity of. CCCP (a mitochondria membrane potential disrupter in DMSO) 10x PBS. Flow cytometry protocol. Cat. No. T3168: M34152 . Cultured human pre-adipocytes loaded with the ratiometric mitochondrial potential indicator JC-1 at 5 µM for 30 minutes at 37°C. In live cells, JC-1 exists either as a green-fluorescent monomer at depolarized membrane potentials or as an orange-fluorescent J-aggregate at.

CCCP-induced parkin-mitochondrial aggregate formation is

A potent uncoupler of mitochondrial oxidative phosphorylation. CCCP is a useful tool for depolarizing mitochodrial membrane potential. Product Specification. Alternative Name: (3-Chlorophenyl)hydrazonomalonitrile : Formula: C 9 H 5 ClN 4 : MW: 204.6 : CAS: 555-60-2 : Purity: ≥98% (TLC) Identity: Determined by IR. Appearance: Yellow-orange solid. Solubility: Soluble in DMSO. Shipping: Ambient. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) is a typical mitochondrial uncoupler, which is widely used in studies on mitochondrial uncoupling. Earlier work in the 1980s showed that CCCP prevented noradrenaline-induced contractions in rabbit mesenteric arteries (Haeusler et al., 1981). A recent study used CCCP to investigate th Mitochondrial permeability transition (MPT) and cytochrome c redistribution from mitochondria are two events associated with apoptosis. We investigated whether an MPT event obligatorily leads to cytochrome c release in vivo. We have previously shown that treatment of human osteosarcoma cells with the protonophore m‐chlorophenylhydrazone (CCCP) for 6 h induces MPT and mitochondrial swelling. CCCP (Carbonyl cyanide m-chlorophenyl hydrazone, Carbonyl cyanide 3-chlorophenylhydrazone), an oxidative phosphorylation inhibitor, is a protonophore mitochondrial uncoupler that increases membrane permeability to protons, leading to a disruption in the mitochondrial membrane potential

Mitochondrial uncoupler carbonyl cyanide m

subunits of the mitochondrial electron transport chain, CCCP treatment has a more robust effect on retarding mitochondrial function as it depolarizes the inner mitochondrial membrane to effectively uncouple oxidative phosphorylation from the electron transport chain. Our results clearly demonstrate tha CCCP is a protonophore which uncouples oxidative phosphorylation, induces ROS and depolarizes mitochondrial membrane potential, thus, triggering mitophagy and bulk autophagy . Researchers have typically challenged cells with CCCP to initiate the autophagy for assessing the oxidative stress induced autophagic flux and mitophagy. Bafilomycin is commonly used to inhibit autophagy by targeting lysosomes. To determine the level of autophagy in MELAS cells, the classical autophagy markers LC3-I. CCCP, an uncoupler and antimycin A, an inhibitor of the mitochondrial electron transport chain, inhibited IL-1β release induced by ATP but not by other stimuli. CCCP did not inhibit the ATP-induced generation of reactive oxygen species and cell death, both of which are known to promote IL-1β release, but did inhibit the ATP-induced activation of caspase-1, a component of the NLRP3 inflammasome. These results suggest that mitochondrial function is required somewhat specifically for ATP. When HeLa cells are treated with CCCP, which depolarizes mitochondria by increasing membrane permeability to H +, a large increase in endogenous full-length PINK1 (∼63 kDa) is seen beginning by 30 min and continuing for at least 3 h

Bcl-2 prevents loss of mitochondria in CCCP-induced

The Mitochondrial Membrane Potential Assay Kit (II) is a fluorescent assay that detects the mitochondrial membrane potential in living cells. The kit includes the cationic dye TMRE (tetramethylrhodamine ethyl ester perchlorate) and a mitochondrial membrane potential disruptor CCCP (carbonyl cyanide 3-chlorophenylhydrazone). TMRE is a cell membrane permeable, fluorescent dye that accumulates in intact mitochondria. Depolarized or inactive mitochondria exhibit decreased membrane potential. After mitochondrial depolarization with carbonyl cyanide m-chlorophenylhydrazone (CCCP), Flag-gp78 induced a threefold loss of depolarized mitochondria and significant loss of the inner mitochondrial protein OxPhosV. Flag-gp78-dependent loss of OxPhosV, but not Mfn1 or Mfn2, was prevented by small interfering RNA (siRNA) knockdown of the autophagy protein Atg5 in CCCP-treated cells. Gp78-induced mitophagy required ubiquitin ligase activity, as it is not observed upon. CCCP was found to reduce mitochondrial membrane potential as seen using JC-1 dye; but interestingly, when menadione was used, mitochondrial membrane potential was not altered . To determine whether CCCP exposure causes mtDNA damage, quantitative alkaline Southern blots were employed over a range of doses of CCCP. CCCP did not produce detectable mtDNA damage in HeLa cells over the range of. The mitochondrial inhibitors reduced the cellular ATP/ADP ratio, however, this would have restrained the ATP dependent Ca2+ buffering processes leading to elevation of [Ca2+]i. In contrast our results showed repression of Ca2+ signal except in case of CCCP which drastically reduced the ATP/ADP ratio. It was inferred that, under the conditions. At 1-h CCCP, 67% of parkin-positive mitochondria were also positive for optineurin, and by 1.5-h CCCP, 84% of parkin-positive mitochondria were also positive for optineurin. By 2 h of CCCP treatment, most mitochondria were positive for both parkin and optineurin (Fig. S5 B-D) and remained so after 6 h of CCCP treatment (Fig. S5 E-H)

  1. e (TMPD) in the presence of.
  2. e whether endogenous Parkin undergoes similar translocation after depolarization, we immunostained cortical neurons at DIV10 with an anti-Parkin antibody following 24-hr CCCP/LIs treatment
  3. Die Atmungskette ist ein Teil des Energiestoffwechsels der meisten Lebewesen.Einerseits wird mit dem Ausdruck Atmungskette ein Stoffwechselweg bezeichnet, nämlich eine Kette von nacheinander stattfindenden biochemischen Redoxreaktionen, die den Lebewesen zur Energiegewinnung dient, andererseits auch die Gesamtheit der an dem Stoffwechselweg teilnehmenden Proteinkomplexe
  4. ) both in the presence and absence of cycloheximide ( Fig. 3 G and not depicted)
  5. 产品说明. Mitochondrial Membrane Potential Assay Kit (II) 是一种检测活细胞线粒体膜电位的荧光检测试剂盒。该试剂盒包含阳离子染料 TMRE(四甲基若丹明乙酯高氯酸盐)和线粒体膜电位分配物 CCCP(羰基氰基-3-氯苯腙)
  6. CS is a quantitative measurement of mitochondrial content, and we have previously shown that loss of CS activity following CCCP treatment can be (a) increased with over‐expression of parkin or (b) impaired by transient KD of PINK1 with siRNA (Gegg et al
  7. Here, we report that both carbonyl cyanide m‐chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%

the mitochondria. Concentrations of CCCP or FCCP in excess of 1 μM, as used by Narendra et al. (9) and by us here, can affect cellular functions other than ΔΨ m (11, 12). Nonetheless, as much as 30-50% of WT Parkin-YFP-transfected cells did show Parkin translocation to mitochondria triggered by mitochondrial depo GABARAPL2 (GABARAPL2-II) were upregulated by carbonyl cyanide 3-chlorophenylhydrazine (CCCP), which induces mitochondrial dysfunction and fragmentation. In contrast, CCCP upregulated the conversion of LC3B-I to LC3B-II, but it did not affect the expression of total LC3B

Mitochondrial biogenesis and degradation are induced by

CCCP is a protonophore mitochondrial uncoupler that increases membrane permeability to protons, leading to a disruption in the mitochondrial membrane potential. 1 It inhibits mitochondrial respiration and ATPase activity when used at concentrations of 110 and 35 nM, respectively. 2 CCCP inhibits activation of stimulation of interferon genes (STING), decreasing the expression of downstream. Mitochondrial dynamics in postmitotic cells regulate neurogenesis Ryohei Iwata 1,2,3,4,5, Pierre Casimir , Pierre Vanderhaeghen1,2,3,4,5* The conversion of neural stem cells into neurons is associated with the remodeling of organelles, but whether and how this is causally linked to fate change is poorly understood. We examined and manipulated mitochondrial dynamics during mouse and human.

We then demonstrated that annexin A7 (ANXA7), a calcium-dependent phospholipid-binding protein, can translocate to impaired mitochondria upon CCCP treatment, where it played a pivotal part in the process of Parkin-dependent mitophagy via interacting with BASP1. As a mitochondrial uncoupling agent, CCCP indirectly regulated ANXA7 and BASP1 to induce Parkin-dependent mitophagy Effect of CCCP treatment on mitochondrial function was still observed after maturation but did not affect parthenogenetic development of oocytes. CCCP-treated oocytes were further cultured for 44 h after which the ATP and ROS contents in mature oocytes and the parthenogenetic developmental rate of oocytes to blastocysts were examined. The difference between the ATP contents of the 10 μM CCCP.

Mitochondrial Membrane Potential Assay Kit (I) is a fluorescent assay that detects the mitochondrial membrane potential in living cells. The kit includes the cationic dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide) and a mitochondrial membrane potential disruptor CCCP (carbonyl cyanide 3-chlorophenylhydrazone). JC-1 is a cell membrane permeable. Combining live fluorescence microscopy and biochemistry, we show that the FBS/BSA in the cell culture medium reduces the ability of CCCP to induce PINK1 accumulation at depolarized mitochondria, subsequent PRKN recruitment and ubiquitin phosphorylation, and ultimately mitochondrial clearance. As a result, high concentrations of CCCP are required to induce mitophagy in FBS/BSA containing media. The mitochondria of cccp‐treated cells are small (C) and those of valinomycin‐treated cells are significantly enlarged (D). Valinomycin‐treated cells show mitochondria enveloped by a continuous outer membrane but containing matrix spaces with different electron densities and cristae morphologies, suggesting that these matrix spaces are separated by unfused inner membrane (arrowheads.

(a) Illustrations of protons movement after CCCP addition

Carbonyl cyanide 3-chlorophenylhydrazone >= 97 % TLC

The mitochondrial inner membrane imports numerous proteins that span it multiple times using the membrane potential Δψ as the only external energy source. We purified the protein insertion complex (TIM22 complex), a twin-pore translocase that mediated the insertion of precursor proteins in a three-step process. After the precursor is tethered to the translocase without losing energy from the. We also treated the protoplasts with carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which is a mitochondrial membrane potential inhibitor, before staining with JC-1. The results showed that the protoplasts treated with CCCP exhibited stronger green signals than the untreated protoplasts (Fig. 5a), suggesting that more apoptotic mitochondria were generated when the protoplasts were treated. f. Les agents découplants (2,4 dinitrophénol, CCCP).. Ils dissipent artificiellement le gradient de protons et il n'y a plus synthèse d'ATP. Le transfert d'électrons et la consommation d'O 2 sont à leur maximum.. Le carbonyl cyanide m-chloro-phenyl hydrazone (CCCP) est un composé lipidique faiblement acide (figure ci-dessous).C'est un agent découplant trés fort M7514), whose fluorescence monitors total mitochondrial mass and in particular, we want to study mitochondrial mass at basal conditions and after the treatment with CCCP. Unexpectedly, we found. 脱共役剤 (だつきょうやくざい、Uncoupler)とは、生物の 酸化的リン酸化 において、 電子伝達系 および ATP 合成反応のいずれをも阻害せずに両反応の共役を阻害する化合物のこと。. 酸化的リン酸化は、電子伝達系により ミトコンドリア 内 膜 内側からくみ出された プロトン が、 ATP合成酵素 を通って流入する際にATPが合成されるという形で両反応が共役し.

Following CCCP treatment, healthy donor MDMs demonstrated marked disruption of the mitochondrial network . When bafilomycin was used in combination with CCCP treatment (to prevent mitophagosome degradation), clusters of mitochondria were found to be surrounded by actin cages, suggesting successful identification of damaged mitochondria and isolation from the healthy network. In some instances. Efavirenz induces neuronal autophagy and mitochondrial alterations. Authors: Purnell J Pharmacol Exp TMRM fluorescence intensity before and after CCCP addition was used for the quantitation of change in mitochondrial membrane potential in spinal cord neurons induced by glutamate receptor agonist AMPA. Tocris Bioscience is the leading supplier of novel and exclusive tools for life science. mCherry-Parkin 200 + CCCP cytosolic mitochondrial 0 20 40 60 80 100 mitochondria-associated Parkin (% cells) GFP-LC3. Supplemental Data Figure S1, Related to Figure 1. Parkin promotes ubiquitination and removal of depolarized mitochondria (A and B) HeLa cells were transfected with a plasmid encoding mCherry-Parkin (400 ng) for 24 h and treated with CCCP (10 µM) for a further 24 h. Cells were.

Congratulations to our neighbors in the Shao lab for the great collaboration with Alban in the lab on the identiifcation of ATP13A1 as a transmembrane dislocase that removes mislocalized tail-anchored proteins from the ER. Such mis-localized proteins include those that normally are localized to mitochondria. Thanks for the great collaboration When mitochondria are damaged, the mitochondrial kinase, PINK1, is stabilized, resulting in the recruitment of the Parkin E3 ligase to damaged mitochondria and ubiquitination of several OMM proteins to trigger mitophagy. Adaptor proteins, such as p62, NBR1, NDP52, Tax1BP1, and optineurin that bind ubiquitin and the autophagosome-associated protein, LC3, may direct the isolation membrane. Mitochondrial dysfunction is a critical factor in the onset and progression of neurodegenerative diseases. Recently, mitochondrial transplantation has been advised as an innovative and attractive strategy to transfer and replace damaged mitochondria. Here we propose, for the first time, to use rat brain extracted synaptosomes, a subcellular fraction of isolated synaptic terminal that contains. Mitochondrial clearance was impaired in reticulocytes derived from Ulk1-deficient animals compared with wild-type littermates (Figure 4B). This was not due to a complete inability to clear mitochondria as this defect could be overcome by culturing in the presence of the mitochondrial uncoupler, CCCP (Figure 4B). It should be noted that.

CCCP-induced changes in the mitochondrial membrane pote

(I) CCCP-uncoupled ('mito CCCP ') or control ('mito DMSO ') mitochondria were incubated for 60 min with 100 nM parkin WT or C431A and in the presence or absence of 2 mg/ml cytosol from parkin-/-mouse brain ('KO cytosol'). Mfn2 and VDAC1 ubiquitination were assayed by immunoblot. Ubiquitinated species are indicated by red asterisks, while unmodified bands are denoted by arrowheads 1. Add Mtphagy Dye working solution BEFORE inducing mitophagy (Mtphagy Dye accumulates in the intact mitochondria) 2. Use appropriate filter - Mtphagy Dye: Ex.500-560 nm, Em.670-730 nm for microscopy detection 3. Use CCCP or FCCP for the positive control *FCCP or CCCP concentration: 10 uM-30 uM, incubation time at 37℃ for 3 hour Bax is not mobilized to mitochondria under these conditions. However, subsequent exposure of CCCP-treated cells to etoposide or staurosporine for 48 h results in rapid cell death and cytochrome c release that is accompanied by Bax association with mitochondria, demonstrating competency of these mitochondria to release cytochrome c with additional triggers. Our findings suggest that MPT is not a sufficient condition, in itself, to effect cytochrome c release channels in the CCCP- and CGP37157-induced actions. CONCLUSIONS AND IMPLICATIONS Mitochondria have a modulatory role on uterine contractions, with mitochondrial inhibition reducing contraction amplitude and pacemaker frequency by changes in Vm, [Ca2+] c and/or Ca 2+ influx. Abbreviations [Ca2+ Using CCCP as uncoupler to depolarize mitochondria and trigger activation of PINK1 and Parkin pathway, as a surrogate to study their function in Parkinson disease Experimental Design and Results Summar

CCCp (50 µM), a mitochondrial membrane potential disrupter, was used as control. The excitation length for JC-1 is 490 nm, and red and green emission lengths are 590 and 529 nm, respectively. The excitation length for JC-1 is 490 nm, and red and green emission lengths are 590 and 529 nm, respectively CCCP is a lipid-soluble weak acid and a potent mitochondrial uncoupling agent that increases the proton permeability across the mitochondrial inner membranes, thus dissipating the transmembrane potential and depolarizing the mitochondria. CCCP could induce the lysosomal removal of depolarized mitochondria (9, 12), which was considered to be mediated by autophagy Then, Vis-A is applied to detect the mitochondrial viscosity of living cells by Fluorescence Lifetime Imaging (FLIM). Especially, with the aid of Vis-A, the changes in viscosity under typical pathological conditions (i.e., treatment with rotenone and carbonylcyanide-m-chlorophenylhydrazone (CCCP)) for mitochondria are monitored by FLIM As CCCP treatment leads to mitochondrial depolarization and reactive oxygen species (ROS) generation, we speculated that both may affect MitoTracker Green staining, with ROS generation induced by CCCP treatment having a more prominent effect on MitoTracker staining than mitochondrial depolarization. The surplus might be responsible for the intensity increase in MitoTracker Green. To test if ROS are able to enhance MitoTracker Green fluorescence, tert-butyl hydroperoxide (TBHP), which is. 3) The positive control is CCCP or FCCP. Briefly, we use CCCP treatment (4 hours, 10 uM) as a positive control to demonstrate collapsed ΔΨm, which is indicated by a clear decrease in MitoTracker.

Membrane depolarization activates the mitochondrial

Mitochondria are involved in many crucial cellular processes. Maintaining healthy mitochondria is essential for cellular homeostasis. Parkin-dependent mitophagy plays an important role in selectively eliminating damaged mitochondria in mammalian cells. However, mechanisms of Parkin-dependent mitophagy remain elusive. In this research, we performed data-independent acquisition-based quantitative mitochondrial proteomics to study the proteomic alterations of carbonyl cyanide m. There has been a growing interest in mitophagy, mitochondria-selective autophagy, which plays an essential role in maintaining intracellular homeostasis. We have developed a small-molecule fluorescent probe, Mtphagy Dye, for visualizing mitophagy, which was readily synthesized from a known perylene derivative, perylene-3,4-dicarboxylic anhydride. Mtphagy Dye has suitable fluorescent properties. Fragmentation of mitochondria was evaluated by live cell imaging ; cleavage of OPA1, a mitochondrial fusion factor cleaved and generated as a short form (S-OPAl) from a long form (L-OPAl) during mitochondrial fission, was detected by western blotting. Results: Mitochondrial fragmentation induced by CCCP was suppressed by propofol. Moreover, cleavage of L-OPAl to S-OPAl was delayed by the. ized with mitochondria, confirming that these two treatments induce PINK1 accumulation. Furthermore, while mitochon-dria in CCCP-treated cells became fragmented as an established consequence of OPA1 degradation by OMA1,26 the mitochon-dria in Dox-treated cells displayed an elongated network, agai F5: Mitochondrial elongation and increased Drp1 S637-PO4 caused by MiD overexpression can be reversed by CCCP. (A) Reduction of Drp1 S637-PO4 levels in CCCP treated cells. Left, lysates from control HeLa cells or HeLa cells expressing MiD49-Myc or MiD51-Myc were analyzed by Western blotting for Drp1

of mitochondrial membrane potential - we utilized the reversible inhibitor of oxidative phosphorylation, CCCP. Low (2.5μM), medium (5μM), and high doses (10μM) of CCCP were selected based on published data using isolated mitochondria showing a dose-dependent Bowling et al. BMC Molecular and Cell Biology (2019) 20:33 Page 2 of 2 m-chlorophenylhydrazone (CCCP), a protonophore used to decrease DJ m.10 In contrast with Mitotrackers relying on DJ m, Tom20 is a constitutive protein of mitochondria, allowing staging of mitochondria by Tom20-GFP irrespective of DJ m.As expected, RC-TPP predominantly resides in mitochondria of control cells whereas CCCP-treated cells exhibit attenuated mitochondrial coumarin uorescence and. Loading mitochondria with AA quenched mitochondrial ROS and inhibited oxidative mitochondrial DNA damage. mtAA inhibited oxidative stress resulting from rote‐none‐induced disruption of the mitochondrial respiratory chain and prevented mitochondrial membrane depolarization in response to a protonophore, CCCP. Our results show that analogous to the cellular uptake, vitamin C enters mitochondria in its oxidized form via Glut1 and protects mitochondria from oxidative injury. Since. a mitochondrial membrane potential disruptor CCCP (carbonyl cyanide 3-chlorophenylhydrazone). TMRE is a cell membrane permeable, fl uorescent dye that accumulates in intact mitochon-dria. Depolarized or inactive mitochondria exhibit decreased membrane potential, resulting in reduced TMRE accumulation. The mean fl uorescence intensity (MFI) of the red emission (561 nm LP) can be used as an. Following CCCP treatment, healthy donor MDMs demonstrated marked disruption of the mitochondrial network . When bafilomycin was used in combination with CCCP treatment (to prevent mitophagosome degradation), clusters of mitochondria were found to be surrounded by actin cages, suggesting successful identification of damaged mitochondria and isolation from the healthy network. In some instances, the network was completely disrupted, with only fragments of mitochondria visible within actin cage

To examine mitochondrial morphology in WT, ρ 0, and CCCP-treated WT lines, cells were immunolabeled for the TOM20 protein, which is located in the mitochondrial outer membrane, providing imaging of the mitochondrial network in these cells. Mitochondrial localization was confirmed by colocalization with MitoTracker (not shown). WT cells visualized by confocal fluorescence microscopy revealed a balance of both fusion and fission, with highly interconnected, reticular mitochondria existing in. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) has been used:. in the inhibition of efflux pump in clinical Pseudomonas aeruginosa.. to inhibit oxidative phosphorylation activity of mitochondria in human Beas-2B bronchial epithelial cells.. to prepare carbonyl cyanide m-chlorophenylhydrazone solution for oxidative and mitochondrial stress assay and mitophagy induction in Caenorhabditis elegans In contrast to Hipk2 +/+ MEF, mitochondria in Hipk2 −/− MEF showed significantly higher basal Parkin protein level in the mitochondria before and after CCCP treatment (Fig. 4J,K). Together, these results support the idea that the loss of HIPK2 results in consistently high Parkin protein levels in the cytosol and mitochondria at basal level and after CCCP-induced mitochondrial membrane. For the mitochondria, the pellets were resuspended in storage buffer and treated with CCCP for 5 min, followed by incubation with 0.1X JC-1 staining solution for 5 min. The dual fluorescence was detected by a mono-photometric system, similar to the protoplasts

CCCP-induced elimination of mitochondria was selective, because peroxisomes were not removed (Fig. 6H) (Narendra et al., 2008). Exposure to valinomycin (1 μ m) for 24 h also triggered Parkin-dependent mitochondrial clearance, similar to CCCP (Fig. 6A,B) CCCP Treated Jurkat Cells. Jurkat cells treated with CCCP (Carbonyl cyanide m-chlorophenyl hydrazone) were stained with JC-1, imaged on the Cellometer Vision CBA and data exported to FCS express. The loss of mitochondrial membrane potential results in the drastic decrease of Mito Red, seen in both the fluorescent image and the generated histogram CCCP directly interferes with mitochondrial function and induces apoptosis. We show that Bcl-2 inhibits apoptosis and that the antiapoptotic effect of Bcl-2 takes place upstream of caspase activation and nuclear changes associated with apoptosis, since these were markedly inhibited in cells overexpressing Bcl-2. Bcl-2 does not prevent the decrease in mitochondrial membrane potential nor the. (C) CCCP reverses mitochondrial elongation in MiD-overexpressing MEFs. Cells were transfected with MiD49-Myc or MiD51-Myc and subsequently treated with vehicle (DMSO) or CCCP. MiD-expressing cells were identified by Myc immunofluorescence, and mitochondria were visualized with Tom20 immunofluorescence. In the DMSO-treated samples, note that MiD-Myc expression causes mitochondrial elongation. In the CCCP-treated samples, note that MiD-Myc-expressing cells have fragmented mitochondria. Scale. a mitochondrial membrane potential disruptor CCCP (carbonyl cyanide 3-chlorophenylhydrazone). TMRE is a cell membrane permeable, fluorescent dye that accumulates in intact mitochon-dria. Depolarized or inactive mitochondria exhibit decreased membrane potential, resulting in reduced TMRE accumulation

The 52-kD form of endogenous PINK1 is found insideVCP Is Essential for Mitochondrial Quality Control by

Mitochondria Function Assays Thermo Fisher Scientific - D

Sep 2020. Congratulations to our neighbors in the Shao lab for the great collaboration with Alban in the lab on the identiifcation of ATP13A1 as a transmembrane dislocase that removes mislocalized tail-anchored proteins from the ER. Such mis-localized proteins include those that normally are localized to mitochondria In this study, we report that the mitochondrial uncoupler CCCP promotes PINK1/PARKIN-mediated mitophagy in myogenic C2C12 cells. As a result of this excess mitophagy, we show that CCCP treatment of myotubes leads to the development of myo-tube atrophy in vitro. Surprisingly, we also found that siRNA-medi- ated knockdown of Parkin results in impaired mitochondrial turnover. In addition. Mitochondrial acidification induced by p7. (A) The mitochondrial samples were stained with 1 µM BCECF-AM for time-course analysis by flow cytometry. (B) Isolated mitochondria were treated with TAMRA-p7 for 5 min and stained with BCECF-AM for 10 min. (C) Experimental data were quantified with flow cytometry. CCCP was used as a positive control. mitochondrial energetics. RNA interference (RNAi) against 17 genes shown to extend lifespan also decrease DeltaPsi(m). Furthermore, lifespan can be significantly extended with the uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP), which dissipates DeltaPsi(m). We conclude that longevity pathways converge on th

-- - +- CCCP -+ 12 34 56 7 Mitochondria P M Import (%) 34 36 MACS DC. Mitochondria Isolation Kit, mouse tissue For 25 isolations from mouse tissue Figure 4: Integrated tissue dissociation followed by mitochondria isolation. The Mitochondria Isolation Kit, mouse tissue is intended for isolation of mitochondria from any mouse tissue such as brain, liver, or skeletal muscle. Tissues can. Thenoyltrifluoroacetone (TTFA), an inhibitor of mitochondrial electron transport chain complex II, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, completely prevented the effects of hyperglycemia, suggesting that reactive oxygen species arise from the mitochondrial electron transport chain. Hyperglycemia potentiated both platelet aggregation and. mitochondrial uncoupler) for 2 h. The CCCP treatment was found to significantly reduce ATP content, increase the amount of phosphorylated AMP-activated protein kinase and elevate reactive oxygenspecies levels in oocytes. When the CCCP-treated COCswere cultured further for 44 h in maturation medium, the ATP levels were restored and the parthenogenetic developmental rate of oocytes to. Moreover, CCCP treatment also reduced mitochondrial transmembrane potential (MTP, ΔΨm) and induced Bax translocation to the mitochondria of its own account. This sensitization was inhibited with N-acetyl-L-cysteine (NAC) treatment by abrogating the ROS which was generated by the combined treatment of CCCP and TRAIL. Of interest, NAC also inhibited reduction of the ΔΨm and Bax translocation. After inducing mitochondrial damage by CCCP for 2 h, mitochondrial fractions were separated from total cell lysates and then analyzed by Western blot (Fig. 7a). Compared to the negative control, miR-27a/b strongly inhibited Parkin translocation to mitochondria as well as accumulation of active form of LC3 (LC3-II), an autophagic marker, upon CCCP treatment (Fig. 7a , b )

Anaerobic respiration coupled with mitochondrial fatty

(vehicle only) and positive (FCCP or CCCP at 2-10 µM) control samples. Incubate the cells in a 5% CO 2, 37 °C incubator. Note: FCCP or CCCP treatment for 15-30 minutes is sufficient to induce apoptosis in most cell lines. The concentration of FCCP or CCCP necessary to induce apoptosis may need to be titrated Figure 3. Confirmation of mitochondrial uncoupling with CCCP in the neurosphere model. Representative blots of OPA1 isoforms. Expected changes from long to short OPA1 isoforms were observed upon uncoupler treatment in all the genotypes, as mitochondria undergo depolarisation/fission following mitochondrial uncoupling (24h, 10 µM CCCP) (A and B.

Temporal integration of mitochondrial stress signals by

Thus, two different types of mitochondrial stresses, MOMP and CCCP, cause mitochondrial depolarization and induce mitophagy. We therefore examined if there is a mechanistic link between two mitophagy pathways. Focusing on the roles of autophagy and apoptosis regulators using isogenic hematopoietic cell lines, our studies demonstrate that MOMP-induced mitophagy is dependent upon Bcl-2 family. Mitochondrial size decreased after CCCP treatment in both Control (Cont A and Cont B) and PARK2 (PA and PB) fibroblasts. Scale bar, 20 μm. (D) iPSCs were treated with 30 μM CCCP or DMSO for 48 h and then stained for CIII coreI (magenta) to label IMM, Oct4 (blue) to label iPSCs, and Hoechst (Ho, white). Mitochondrial size in Control (Cont A (B7), Cont B (WD39)), and PARK2 (PA9 and PB2) iPSCs. Description: Oxidative phosphorylation uncoupler. Chemical Name: Carbonyl cyanide 3-chlorophenylhydrazone. Purity: ≥98%. Product Details. Citations (4) Reviews (1 CCCP treatment also results in PINK1 stabilization on the mitochondrial membrane, which subsequently increases Parkin recruitment from the cytosol to abnormal mitochondria. In addition, we found that CCCP increased the level of mitochondrial LC3II, indicating that Parkin recruitment of PINK1 is a result of mitophagy. We propose that activation of PGAM5 is associated with DRP1 recruitment and. Depolarization of mitochondria by the protonophore CCCP is a strategy increasingly used to experimentally trigger not only mitophagy, but also bulk autophagy. Using live-cell fluorescence microscopy we found that treatment of HeLa cells with CCCP caused redistribution of mitochondrially targeted dyes, including DiOC6, TMRM, MTR, and MTG, from mitochondria to the cytosol, and subsequently to.

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