cytochrome c Search Results


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  • 97
    Thermo Fisher cytochrome c
    Effect of chrysin on <t>cytochrome</t> c and caspase 3 protein expressions and activity in rats subjected to chronic doxorubicin (DOX) intoxication. ( A ) Protein expression of cytochrome c by immunohistochemical staining. ( B ) Protein expression of caspase 3 by immunohistochemical staining. Scale bar, 50 µm (200x) and quantitative image analysis for immunohistochemical staining was expressed as optical densities (OD) across 10 different fields for each rat section. ( C ) Caspase 3 activity expressed as pmolpNA/min/mg protein. Data are represented as as mean ± SD (n = 10). a or b: Statistically significant from the control or DOX group respectively at P
    Cytochrome C, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 895 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Millipore cytochrome c
    Dose–response curves of retrograde transport of neurotrophins and <t>cytochrome</t> C from the eye to the ION in 15-d-old chick embryos ( A ) and hatchling chicks ( B ). The average number of autoradiographic grains/ION neuron is plotted as a function of the amount in the injected eye at the time the animal is killed. A , Average specific activities for these experiments were 126.6 cpm/pg (NT-3), 125.5 cpm/pg (BDNF), 74.8 cpm/pg (NGF), and 57.9 cpm/pg (Cyt. C). BDNF and NT-3 are transported with significantly higher efficiencies than NGF in the lower dose range, but not at higher doses. These differences are not attributable to differences in the specific activities, because grain densities correlate in a linear fashion with the amount of radioactivity up to 30 grains/neuron (data not shown). Each data point is the mean of three to nine experiments. Error bars = SEM. B , Retrograde transport of 125 I-labeled neurotrophins from the eye to the ION is reduced significantly in hatchlings (P1, black symbols ) compared with 15-d-old embryos (E15, open symbols ). Open symbols: averages; black symbols: values from individual experiments (NT-3, n = 5; BDNF, n = 4; NGF, n = 2).
    Cytochrome C, supplied by Millipore, used in various techniques. Bioz Stars score: 80/100, based on 4979 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc cytochrome c
    Effects of NaHS on mitochondrial function. ( A ) ATP synthesis. After pretreatment with vehicle, 300 μM NaHS or 10 mM PAG for 6 hours and followed by exposure to 600 μM H 2 O 2 for another 4 hours, HUVECs were harvested to collect mitochondria. The rate of ATP synthesis was expressed by µmol ATP/min/g of mitochondrial protein. ( B ) Release of <t>cytochrome</t> c from mitochondria. After treatments as previous description, HUVECs were harvested to collect mitochondria and cytosol. The protein expression was tested by western blot. The bar chart showed the ratio of cytochrome c in cytosol to that in mitochondria, indicating the intensity of release of cytochrome c. ( C ) MDA changes in HUVECs mediated by H 2 O 2 . The data are expressed at nmol/mg. ( D ) Fluorescent intensity of DPPP in HUVECs mediated by H 2 O 2 . ( E ) ROS production was stained by 10 μM H 2 DCFDA for 20 min, whose oxidation product (DCF) fluorescence indicated ROS formation. ( F ) ROS production was stained by 5 μM DHE for 30 min, which fluorescence indicated ROS formation. The absorbance values in (D)–(F) of HUVECs were normalized against the values for normal controls and expressed as a percentage of control. ( G )–( L ) JC-1 staining. Red fluorescence represents the mitochondrial aggregate form of JC-1, indicating intact mitochondrial membrane potential. Green fluorescence represents the monomeric form of JC-1, indicating dissipation of Δ Ψ m . (G)–(J) HUVECs were stained with JC-1. (K) CCCP was the positive control. Cells were observed under ×200 microscopy. Scale bar is shown at 100 μm. (L) Ratio of red to green fluorescence, indicating ratio of JC-1 polymer/monomer. The data shown are mean ± SEM (n = 6). * p
    Cytochrome C, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 3894 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology cytochrome c
    Mithramycin reduces etoposide-induced mitochondrial damage and attenuates p53-dependent transcription. RCNs were treated with 50 µM etoposide +/−200 nM Mithramycin for all panels. a The cytosolic fraction was collected after 6h and 24 h. Controls were collected at 24h. Equal amounts of fraction lysates were loaded onto an SDS-polyacrylamide gel and after electrophoretic separation and transfer to a membrane were incubated with antibodies against AIF and <t>cytochrome</t> C proteins. Protein levels (of bands indicated by arrows) were quantified by densitometry, normalized to β-actin signal and are presented as normalized fold change compared with control levels. n = 3/group for all groups. b Cytoplasmic (C), total (T), and nuclear/mitochondrial (N/M) fractions were pooled and probed for cytochrome c oxidase IV (COX IV) and Lamin to confirm purity of cytoplasmic fraction from mitochondria and nuclei, respectively. c 6h after treatments, cellular respiration was measured using a Seahorse XF24 Extracellular Flux Analyzer, and a representative measurement is shown here. Sequential addition of oligomycin, DNP, pyruvate and antimycin A was utilized to identify maximum and spare respiratory capacity ( c ). Each group contains n = 4 averages from separate experiments on different days, each experiment except one contained n = 4+ separately cultured wells of neurons per group (that experiment contained wells that were eliminated due to no significant increase in OCR over baseline indicating a failed injection/port, n = 3+/group for that experiment). d , f – h After 1, 6, or 24h, cells were harvested. Equal amounts of purified RNA were converted into cDNA. Equal volumes of cDNA were loaded for qPCR. mRNA/miRNA levels were normalized via U6/GAPDH (respectively), quantified using the ddCt method and are presented as fold change compared with control levels. n = 3/group for all groups. e Chromatin Immunoprecipitation was done using p53 or Sp1 antibodies, and equal volumes of resulting DNA fragments were loaded for qPCR. Pulled-down DNA levels were normalized using ChIP Inputs, quantified using the 2 −ddCt method and are presented as fold change compared with control levels. f Electrophoresis and western blotting were performed for Sp1 protein. Protein levels were quantified by densitometry, normalized to β-actin signal and are presented as normalized fold change compared with control levels. n = 3/group for all groups. Data all represent mean±SD. Significance assigned based on one-way ANOVA and Tukey post hoc test; * p
    Cytochrome C, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 3154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson cytochrome c
    The combination of cisplatin and mdivi-1 enhances MOMP bypassing Bax/Bak-dependent mechanism (A) Bax/Bak wild-type (Bax/Bak +/+) and double knockout (Bax/Bak −/−) SV40-transformed MEF cells were treated with cisplatin at indicated concentrations for 20 h The cleavage of caspase-9 and -3 were detected by western blot. (B, C) Cleavage of caspase-9 and -3 in Bax/Bak wild-type and double knockout MEF cells after combination treatment. (D) Quantification of apoptotic cells by Annexin V and PI after 8 h treatment of combination at 50 μM. (E) Release of <t>cytochrome</t> c from Bax/Bak wild-type and double knockout MEF cells after combination treatment. These data represent three independent experiments.
    Cytochrome C, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2750 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam cytochrome c
    Expression levels of apoptotic proteins after SIRT1 silencing following ICH.  (A)  Representative western blots of AIF, cytochrome c, caspase-3, and cleaved caspase-3. Relative protein band density values were calculated as the ratio of the protein of interest to that of GAPDH. Quantification of  (A)  is shown in  (B) . Error bars represent mean ± SEM. ( ∗ P
    Cytochrome C, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1408 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti cytochrome c
    The effect of SSB consumption on calpain and <t>cytochrome</t> c expression. ( A ) Calpain and ( B ) cytochrome c levels. Data are displayed as mean ± SEM ( n = 8).
    Anti Cytochrome C, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 845 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson mouse anti cytochrome c
    Bax translocation, <t>cytochrome</t> c release, and cell death following PV infection depend on JNK activation. (A) Inhibition of JNK activity during PV infection in IMR5 cells treated with SP600125 (25 μM). Cells were incubated with the JNK inhibitor for 2 h before PV infection, and the inhibitor concentration was maintained during the adsorption period and throughout PV infection. Levels of phospho (Thr183/Tyr185)-JNK and phospho (Ser63)-c-Jun in whole-cell lysates were determined at 30 min p.i. by Western blotting. Blots were subsequently stripped and reprobed with antibodies recognizing all JNK and c-Jun forms to confirm equal protein loading. (B) IMR5 cells were uninfected or were infected with PV in the presence or absence of SP600125 (25 μM). Whole-cell lysates (8 h p.i.) were subjected to Western blot analysis with anti-Bax and anti-cytochrome c (Cyt c) antibodies. Actin was used as a control for protein loading. (C) Inhibition of Bax translocation from the cytosol to mitochondria by the JNK inhibitor SP600125. IMR5 cells were uninfected or were infected with PV in the presence or the absence of SP600125 (25 μM). Eight hours p.i., the cytosolic and heavy membrane fractions were assayed for Bax by Western blotting. Actin and Cox IV were used as controls for protein loading of cytosolic and heavy membrane fractions, respectively. Protein levels of heavy membrane and cytosolic fractions were determined by densitometry and plotted as ratios relative to the levels of Cox IV and actin, respectively. (D) Cytochrome c release is reduced by JNK inhibitor. IMR5 cells were uninfected or were infected with PV in the presence or absence of SP600125 (25 μM). Cytosolic extract proteins were analyzed at 14 h p.i. by immunoblotting with anti-cytochrome c (Cyt c) antibody. Actin was used as a control for protein loading. Protein levels were determined by densitometry and plotted as ratios relative to the levels of actin. (E) Inhibition of PV-induced cytopathic effect by the JNK inhibitor SP600125. Cells were uninfected or were infected with PV in the presence or absence of SP600125 (25 μM) and visualized by light microscopy at the indicated times p.i. Magnification, ×350. (F) The JNK inhibitor SP600125 does not affect PV growth but affects PV release. IMR5 cells were infected with PV in the presence or absence of SP600125 (25 μM). Total virus yield (extracellular and intracellular) was determined by TCID 50 assay at the indicated times after three cycles of freezing and thawing to release intracellular viruses. The titers of extracellular virus were determined from the supernatant of PV-infected cells at the indicated times after the removal of detached cells by centrifugation. Each point represents the mean virus titer for two independent experiments. Standard errors of the means are indicated. *, P
    Mouse Anti Cytochrome C, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 704 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore horse heart cytochrome c
    Hydrodynamic radius ( a ) and electric dipole moment ( b ) of <t>cytochrome-c</t> in water/ethylene-glycol mixtures as function of weight fraction of cosolvent. The error bars are the result of a statistical analysis connected to the best fit procedure employed to analyze the experimental data. Level of confidence 95%. Dotted lines for visual aim.
    Horse Heart Cytochrome C, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 512 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmingen cytochrome c
    Caspase independent cell death MCF7 cells were exposed to air, hypoxia (Hx) or hypoxia with Baf1A for 36 hrs and the cells fractionated into soluble (cytoplasmic) and heavy membrane (mitochondrial) fractions. Cytoplasmic levels of <t>cytochrome</t> c is shown in (A). Fractional purity was determined by the mitochondrial marker VDAC. In (B) caspase 3 enzymatic activity was determined in MCF7 cells exposed to air (48 hrs) or to hypoxia (Hx) in the presence and absence of Baf1A for times indicated. The level of cleaved ICAD, a target of caspase 3, was determined in hypoxic MCF7 cells exposed to increasing concentrations of Baf1A (C). As a positive control a parallel plate was treated with 1 μM staurosporine, a potent inducer of caspase dependent apoptosis. In (D), proteolysis of the calpain target, α-fodrin, was determined in MCF7 cell exposed to hypoxia and hypoxia-Baf1A for indicated times. Data are means ± SEM. All results are representative of at least 3 experiments.
    Cytochrome C, supplied by Pharmingen, used in various techniques. Bioz Stars score: 92/100, based on 535 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti cytochrome c
    D-gal treatment increases mitochondrial <t>cytochrome</t> c release and cleaved caspase-3 in the auditory cortex of rats. (A) Western blot analysis of Cyt c levels in the mitochondrial fraction. (B) Western blot analysis of Cyt c levels in the cytosolic fraction. (C) Western blot analysis of cleaved caspase-3 levels. The relative levels of Mito Cyt c were normalized to VDAC1; the relative levels of Cyto c and cleaved caspase-3 in each group were normalized to β-actin. * P
    Anti Cytochrome C, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 518 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore cytochrome c oxidase assay kit
    TM treatment does not alter copper-dependent enzyme activity. Results are mean enzyme activity ± SE in brain homogenates from the prevention cohort for (a) <t>cytochrome</t> c oxidase (cytocox) and (b) superoxide dismutase (SOD). wt veh = wild type mice
    Cytochrome C Oxidase Assay Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 430 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology anti cytochrome c
    Schematic representation of the plausible molecular mechanism proposed for the induction of apoptosis by maslinic acid in HT29 colon-cancer cells . This molecular mechanism is regulated via the induction of JNK and p53, resulting in mitochondrial disruption, the release of <t>cytochrome-c</t> and finally the activation of a cascade of caspases.
    Anti Cytochrome C, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 883 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    SMAC Corp cytochrome c
    The current model for the potentiation of TRAIL-induced apoptosis in cancer cells by depolarization . Triggering of TRAIL-R1 (DR4)/TRAIL-R2 (DR5) induces the generation and accumulation of mROS, leading to impairment of the ETC function and cardiolipin oxidation. Impairment of the ETC complex I/III function decreases H + efflux, thereby causing ΔΨ m dissipation, i.e., MMD, and additional mROS generation and cardiolipin oxidation, thereby forming a positive loop. Cardiolipin oxidation and ΔΨ m dissipation cooperatively promote the MPT and liberation of sufficient <t>cytochrome</t> c to trigger caspase activation and induce apoptosis. TRAIL-resistant cancer cells appear to gain considerable tolerance for oxidative stress-mediated activation of the intrinsic death pathway. Accumulation of mROS can also promote the formation of unfolded or misfolded proteins, thereby provoking ERS responses such as activation of the transcriptional factor XBP-1. XBP-1 activation leads to upregulation of the surface TRAIL-R2 expression level, thereby enhancing the death signaling. Activation of this alternative death pathway may contribute to commit TRAIL-resistant cancer cells to apoptosis.
    Cytochrome C, supplied by SMAC Corp, used in various techniques. Bioz Stars score: 93/100, based on 303 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cyt c  (Abcam)
    99
    Abcam cyt c
    IL-2 activated the mitochondrial apoptotic pathway in the presence of sorafenib. a , b Mitochondrial ROS production was detected in HepG2 cells. IL-2 (5 ng/ml) treatment was carried out in the presence of 5 μM sorafenib. c – e The antioxidants in HepG2 cells under IL-2 and sorafenib co-treatment were measured via ELISA. f The mPTP opening rate was analysed to determine the mitochondrial damage. IL-2 (5 ng/ml) treatment was carried out in the presence of 5 μM sorafenib. g , h <t>Cyt-c</t> liberation was observed via immunofluorescence. i – o Mitochondrial apoptotic proteins were analysed by western blotting. The sorafenib-mediated upregulation of apoptotic proteins was further augmented by IL-2 treatment. *P
    Cyt C, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 599 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Cell Signaling Technology Inc cytochrome c oxidase subunit iv
    AS-IV inhibited Aβ1-42-induced <t>cytochrome</t> c release from mitochondria in SK-N-SH cells. A. A representative blots of immunoreactive bands for cytochrome c in cytosol. B. Data were expressed as fold-increase of cytochrome c relative to vehicle. Protein expression levels were normalized to β-actin. C. A representative blots of immunoreactive bands for cytochrome c in mitochondria. D. Data were expressed as fold-increase of cytochrome c relative to vehicle. Protein expression levels were normalized to COX IV. # P
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    94
    Millipore bovine heart
    AS-IV inhibited Aβ1-42-induced <t>cytochrome</t> c release from mitochondria in SK-N-SH cells. A. A representative blots of immunoreactive bands for cytochrome c in cytosol. B. Data were expressed as fold-increase of cytochrome c relative to vehicle. Protein expression levels were normalized to β-actin. C. A representative blots of immunoreactive bands for cytochrome c in mitochondria. D. Data were expressed as fold-increase of cytochrome c relative to vehicle. Protein expression levels were normalized to COX IV. # P
    Bovine Heart, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 438 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam anti cytochrome c antibody
    Colocalization of <t>cytochrome</t> c and mitochondria in blastocysts cultured for 7 days in the absence or presence of E-64. (A) Blastocysts were labeled with a cytochrome c -specific antibody (green) and MitoTracker Red (red). (B) Colocalization was analyzed using Image-Pro Plus, Arrow and frame, released cytochrome c .
    Anti Cytochrome C Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 257 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore equine cytochrome c
    Structures of receptors and proteins (together with relevant properties) described in this work (a) highly functionalized ruthenium tris-bipyridine receptors 1–3 (b) <t>cytochrome</t> c (c) 60% acetylated cytochrome c (d) lysozyme (PDB
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    89
    Cytox perinatal iron deficiency decreases cytochrome c oxidase
    Structures of receptors and proteins (together with relevant properties) described in this work (a) highly functionalized ruthenium tris-bipyridine receptors 1–3 (b) <t>cytochrome</t> c (c) 60% acetylated cytochrome c (d) lysozyme (PDB
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    Image Search Results


    Effect of chrysin on cytochrome c and caspase 3 protein expressions and activity in rats subjected to chronic doxorubicin (DOX) intoxication. ( A ) Protein expression of cytochrome c by immunohistochemical staining. ( B ) Protein expression of caspase 3 by immunohistochemical staining. Scale bar, 50 µm (200x) and quantitative image analysis for immunohistochemical staining was expressed as optical densities (OD) across 10 different fields for each rat section. ( C ) Caspase 3 activity expressed as pmolpNA/min/mg protein. Data are represented as as mean ± SD (n = 10). a or b: Statistically significant from the control or DOX group respectively at P

    Journal: Scientific Reports

    Article Title: Mechanistic clues to the protective effect of chrysin against doxorubicin-induced cardiomyopathy: Plausible roles of p53, MAPK and AKT pathways

    doi: 10.1038/s41598-017-05005-9

    Figure Lengend Snippet: Effect of chrysin on cytochrome c and caspase 3 protein expressions and activity in rats subjected to chronic doxorubicin (DOX) intoxication. ( A ) Protein expression of cytochrome c by immunohistochemical staining. ( B ) Protein expression of caspase 3 by immunohistochemical staining. Scale bar, 50 µm (200x) and quantitative image analysis for immunohistochemical staining was expressed as optical densities (OD) across 10 different fields for each rat section. ( C ) Caspase 3 activity expressed as pmolpNA/min/mg protein. Data are represented as as mean ± SD (n = 10). a or b: Statistically significant from the control or DOX group respectively at P

    Article Snippet: Anti NF-κB p65, VEGF, p53, cytochrome c, caspase 3 antibodies were purchased from Thermo Fisher Scientific Co. (Waltham, MA, USA).

    Techniques: Activity Assay, Expressing, Immunohistochemistry, Staining

    Dose–response curves of retrograde transport of neurotrophins and cytochrome C from the eye to the ION in 15-d-old chick embryos ( A ) and hatchling chicks ( B ). The average number of autoradiographic grains/ION neuron is plotted as a function of the amount in the injected eye at the time the animal is killed. A , Average specific activities for these experiments were 126.6 cpm/pg (NT-3), 125.5 cpm/pg (BDNF), 74.8 cpm/pg (NGF), and 57.9 cpm/pg (Cyt. C). BDNF and NT-3 are transported with significantly higher efficiencies than NGF in the lower dose range, but not at higher doses. These differences are not attributable to differences in the specific activities, because grain densities correlate in a linear fashion with the amount of radioactivity up to 30 grains/neuron (data not shown). Each data point is the mean of three to nine experiments. Error bars = SEM. B , Retrograde transport of 125 I-labeled neurotrophins from the eye to the ION is reduced significantly in hatchlings (P1, black symbols ) compared with 15-d-old embryos (E15, open symbols ). Open symbols: averages; black symbols: values from individual experiments (NT-3, n = 5; BDNF, n = 4; NGF, n = 2).

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Retrograde Transport of Neurotrophins from the Eye to the Brain in Chick Embryos: Roles of the p75NTR and trkB Receptors

    doi:

    Figure Lengend Snippet: Dose–response curves of retrograde transport of neurotrophins and cytochrome C from the eye to the ION in 15-d-old chick embryos ( A ) and hatchling chicks ( B ). The average number of autoradiographic grains/ION neuron is plotted as a function of the amount in the injected eye at the time the animal is killed. A , Average specific activities for these experiments were 126.6 cpm/pg (NT-3), 125.5 cpm/pg (BDNF), 74.8 cpm/pg (NGF), and 57.9 cpm/pg (Cyt. C). BDNF and NT-3 are transported with significantly higher efficiencies than NGF in the lower dose range, but not at higher doses. These differences are not attributable to differences in the specific activities, because grain densities correlate in a linear fashion with the amount of radioactivity up to 30 grains/neuron (data not shown). Each data point is the mean of three to nine experiments. Error bars = SEM. B , Retrograde transport of 125 I-labeled neurotrophins from the eye to the ION is reduced significantly in hatchlings (P1, black symbols ) compared with 15-d-old embryos (E15, open symbols ). Open symbols: averages; black symbols: values from individual experiments (NT-3, n = 5; BDNF, n = 4; NGF, n = 2).

    Article Snippet: Control rabbit IgG and Fab were obtained from Jackson ImmunoResearch Labs (West Grove, PA), K252a from Kamiya Biomedical Company (Thousand Oaks, CA), and cytochrome C (from chicken heart) and monensin from Sigma (St. Louis, MO).

    Techniques: Injection, Radioactivity, Labeling

    Retrograde transport of 125 I-labeled neurotrophins and cytochrome C ( cyt. C ) from the eye to the ION in 15-d-old chick embryos (E15). The upper panels show dark-field images of the ION; the lower panels show bright-field views at higher magnification. Comparable amounts (~60 ng) of radioiodinated NGF ( A ), BDNF ( B ), NT-3 ( C ), or cyt. C ( D ) were injected into the eye with doses of 17–20 ng remaining in the eye at the time animals were killed (= 20 hr after injection). Sections were processed for autoradiography. Note the robust transport of BDNF and NT-3 ( B , C ), compared with the weak transport of NGF ( A ) and faint transport of cyt. C ( D ). Several labeled ectopic ION neurons are visible in the upper panel of B . Scale bars: ( upper ) 200 μ m; ( lower ) 10 μ m.

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Retrograde Transport of Neurotrophins from the Eye to the Brain in Chick Embryos: Roles of the p75NTR and trkB Receptors

    doi:

    Figure Lengend Snippet: Retrograde transport of 125 I-labeled neurotrophins and cytochrome C ( cyt. C ) from the eye to the ION in 15-d-old chick embryos (E15). The upper panels show dark-field images of the ION; the lower panels show bright-field views at higher magnification. Comparable amounts (~60 ng) of radioiodinated NGF ( A ), BDNF ( B ), NT-3 ( C ), or cyt. C ( D ) were injected into the eye with doses of 17–20 ng remaining in the eye at the time animals were killed (= 20 hr after injection). Sections were processed for autoradiography. Note the robust transport of BDNF and NT-3 ( B , C ), compared with the weak transport of NGF ( A ) and faint transport of cyt. C ( D ). Several labeled ectopic ION neurons are visible in the upper panel of B . Scale bars: ( upper ) 200 μ m; ( lower ) 10 μ m.

    Article Snippet: Control rabbit IgG and Fab were obtained from Jackson ImmunoResearch Labs (West Grove, PA), K252a from Kamiya Biomedical Company (Thousand Oaks, CA), and cytochrome C (from chicken heart) and monensin from Sigma (St. Louis, MO).

    Techniques: Labeling, Injection, Autoradiography

    Effects of NaHS on mitochondrial function. ( A ) ATP synthesis. After pretreatment with vehicle, 300 μM NaHS or 10 mM PAG for 6 hours and followed by exposure to 600 μM H 2 O 2 for another 4 hours, HUVECs were harvested to collect mitochondria. The rate of ATP synthesis was expressed by µmol ATP/min/g of mitochondrial protein. ( B ) Release of cytochrome c from mitochondria. After treatments as previous description, HUVECs were harvested to collect mitochondria and cytosol. The protein expression was tested by western blot. The bar chart showed the ratio of cytochrome c in cytosol to that in mitochondria, indicating the intensity of release of cytochrome c. ( C ) MDA changes in HUVECs mediated by H 2 O 2 . The data are expressed at nmol/mg. ( D ) Fluorescent intensity of DPPP in HUVECs mediated by H 2 O 2 . ( E ) ROS production was stained by 10 μM H 2 DCFDA for 20 min, whose oxidation product (DCF) fluorescence indicated ROS formation. ( F ) ROS production was stained by 5 μM DHE for 30 min, which fluorescence indicated ROS formation. The absorbance values in (D)–(F) of HUVECs were normalized against the values for normal controls and expressed as a percentage of control. ( G )–( L ) JC-1 staining. Red fluorescence represents the mitochondrial aggregate form of JC-1, indicating intact mitochondrial membrane potential. Green fluorescence represents the monomeric form of JC-1, indicating dissipation of Δ Ψ m . (G)–(J) HUVECs were stained with JC-1. (K) CCCP was the positive control. Cells were observed under ×200 microscopy. Scale bar is shown at 100 μm. (L) Ratio of red to green fluorescence, indicating ratio of JC-1 polymer/monomer. The data shown are mean ± SEM (n = 6). * p

    Journal: PLoS ONE

    Article Title: Hydrogen Sulfide Protects HUVECs against Hydrogen Peroxide Induced Mitochondrial Dysfunction and Oxidative Stress

    doi: 10.1371/journal.pone.0053147

    Figure Lengend Snippet: Effects of NaHS on mitochondrial function. ( A ) ATP synthesis. After pretreatment with vehicle, 300 μM NaHS or 10 mM PAG for 6 hours and followed by exposure to 600 μM H 2 O 2 for another 4 hours, HUVECs were harvested to collect mitochondria. The rate of ATP synthesis was expressed by µmol ATP/min/g of mitochondrial protein. ( B ) Release of cytochrome c from mitochondria. After treatments as previous description, HUVECs were harvested to collect mitochondria and cytosol. The protein expression was tested by western blot. The bar chart showed the ratio of cytochrome c in cytosol to that in mitochondria, indicating the intensity of release of cytochrome c. ( C ) MDA changes in HUVECs mediated by H 2 O 2 . The data are expressed at nmol/mg. ( D ) Fluorescent intensity of DPPP in HUVECs mediated by H 2 O 2 . ( E ) ROS production was stained by 10 μM H 2 DCFDA for 20 min, whose oxidation product (DCF) fluorescence indicated ROS formation. ( F ) ROS production was stained by 5 μM DHE for 30 min, which fluorescence indicated ROS formation. The absorbance values in (D)–(F) of HUVECs were normalized against the values for normal controls and expressed as a percentage of control. ( G )–( L ) JC-1 staining. Red fluorescence represents the mitochondrial aggregate form of JC-1, indicating intact mitochondrial membrane potential. Green fluorescence represents the monomeric form of JC-1, indicating dissipation of Δ Ψ m . (G)–(J) HUVECs were stained with JC-1. (K) CCCP was the positive control. Cells were observed under ×200 microscopy. Scale bar is shown at 100 μm. (L) Ratio of red to green fluorescence, indicating ratio of JC-1 polymer/monomer. The data shown are mean ± SEM (n = 6). * p

    Article Snippet: The presence of cytochrome c was detected from mitochondrial and cytosol extractions by immunoblot analysis using anti-cytochrome c antibody (1∶1000, Cell Signaling).

    Techniques: Expressing, Western Blot, Multiple Displacement Amplification, Staining, Fluorescence, Positive Control, Microscopy

    Mithramycin reduces etoposide-induced mitochondrial damage and attenuates p53-dependent transcription. RCNs were treated with 50 µM etoposide +/−200 nM Mithramycin for all panels. a The cytosolic fraction was collected after 6h and 24 h. Controls were collected at 24h. Equal amounts of fraction lysates were loaded onto an SDS-polyacrylamide gel and after electrophoretic separation and transfer to a membrane were incubated with antibodies against AIF and cytochrome C proteins. Protein levels (of bands indicated by arrows) were quantified by densitometry, normalized to β-actin signal and are presented as normalized fold change compared with control levels. n = 3/group for all groups. b Cytoplasmic (C), total (T), and nuclear/mitochondrial (N/M) fractions were pooled and probed for cytochrome c oxidase IV (COX IV) and Lamin to confirm purity of cytoplasmic fraction from mitochondria and nuclei, respectively. c 6h after treatments, cellular respiration was measured using a Seahorse XF24 Extracellular Flux Analyzer, and a representative measurement is shown here. Sequential addition of oligomycin, DNP, pyruvate and antimycin A was utilized to identify maximum and spare respiratory capacity ( c ). Each group contains n = 4 averages from separate experiments on different days, each experiment except one contained n = 4+ separately cultured wells of neurons per group (that experiment contained wells that were eliminated due to no significant increase in OCR over baseline indicating a failed injection/port, n = 3+/group for that experiment). d , f – h After 1, 6, or 24h, cells were harvested. Equal amounts of purified RNA were converted into cDNA. Equal volumes of cDNA were loaded for qPCR. mRNA/miRNA levels were normalized via U6/GAPDH (respectively), quantified using the ddCt method and are presented as fold change compared with control levels. n = 3/group for all groups. e Chromatin Immunoprecipitation was done using p53 or Sp1 antibodies, and equal volumes of resulting DNA fragments were loaded for qPCR. Pulled-down DNA levels were normalized using ChIP Inputs, quantified using the 2 −ddCt method and are presented as fold change compared with control levels. f Electrophoresis and western blotting were performed for Sp1 protein. Protein levels were quantified by densitometry, normalized to β-actin signal and are presented as normalized fold change compared with control levels. n = 3/group for all groups. Data all represent mean±SD. Significance assigned based on one-way ANOVA and Tukey post hoc test; * p

    Journal: Cell Death & Disease

    Article Title: Mithramycin selectively attenuates DNA-damage-induced neuronal cell death

    doi: 10.1038/s41419-020-02774-6

    Figure Lengend Snippet: Mithramycin reduces etoposide-induced mitochondrial damage and attenuates p53-dependent transcription. RCNs were treated with 50 µM etoposide +/−200 nM Mithramycin for all panels. a The cytosolic fraction was collected after 6h and 24 h. Controls were collected at 24h. Equal amounts of fraction lysates were loaded onto an SDS-polyacrylamide gel and after electrophoretic separation and transfer to a membrane were incubated with antibodies against AIF and cytochrome C proteins. Protein levels (of bands indicated by arrows) were quantified by densitometry, normalized to β-actin signal and are presented as normalized fold change compared with control levels. n = 3/group for all groups. b Cytoplasmic (C), total (T), and nuclear/mitochondrial (N/M) fractions were pooled and probed for cytochrome c oxidase IV (COX IV) and Lamin to confirm purity of cytoplasmic fraction from mitochondria and nuclei, respectively. c 6h after treatments, cellular respiration was measured using a Seahorse XF24 Extracellular Flux Analyzer, and a representative measurement is shown here. Sequential addition of oligomycin, DNP, pyruvate and antimycin A was utilized to identify maximum and spare respiratory capacity ( c ). Each group contains n = 4 averages from separate experiments on different days, each experiment except one contained n = 4+ separately cultured wells of neurons per group (that experiment contained wells that were eliminated due to no significant increase in OCR over baseline indicating a failed injection/port, n = 3+/group for that experiment). d , f – h After 1, 6, or 24h, cells were harvested. Equal amounts of purified RNA were converted into cDNA. Equal volumes of cDNA were loaded for qPCR. mRNA/miRNA levels were normalized via U6/GAPDH (respectively), quantified using the ddCt method and are presented as fold change compared with control levels. n = 3/group for all groups. e Chromatin Immunoprecipitation was done using p53 or Sp1 antibodies, and equal volumes of resulting DNA fragments were loaded for qPCR. Pulled-down DNA levels were normalized using ChIP Inputs, quantified using the 2 −ddCt method and are presented as fold change compared with control levels. f Electrophoresis and western blotting were performed for Sp1 protein. Protein levels were quantified by densitometry, normalized to β-actin signal and are presented as normalized fold change compared with control levels. n = 3/group for all groups. Data all represent mean±SD. Significance assigned based on one-way ANOVA and Tukey post hoc test; * p

    Article Snippet: Santa Cruz (Dallas, TX): cytochrome c (sc-13560).

    Techniques: Incubation, Cell Culture, Injection, Purification, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Electrophoresis, Western Blot

    The combination of cisplatin and mdivi-1 enhances MOMP bypassing Bax/Bak-dependent mechanism (A) Bax/Bak wild-type (Bax/Bak +/+) and double knockout (Bax/Bak −/−) SV40-transformed MEF cells were treated with cisplatin at indicated concentrations for 20 h The cleavage of caspase-9 and -3 were detected by western blot. (B, C) Cleavage of caspase-9 and -3 in Bax/Bak wild-type and double knockout MEF cells after combination treatment. (D) Quantification of apoptotic cells by Annexin V and PI after 8 h treatment of combination at 50 μM. (E) Release of cytochrome c from Bax/Bak wild-type and double knockout MEF cells after combination treatment. These data represent three independent experiments.

    Journal: Oncotarget

    Article Title: Novel combination of mitochondrial division inhibitor 1 (mdivi-1) and platinum agents produces synergistic pro-apoptotic effect in drug resistant tumor cells

    doi:

    Figure Lengend Snippet: The combination of cisplatin and mdivi-1 enhances MOMP bypassing Bax/Bak-dependent mechanism (A) Bax/Bak wild-type (Bax/Bak +/+) and double knockout (Bax/Bak −/−) SV40-transformed MEF cells were treated with cisplatin at indicated concentrations for 20 h The cleavage of caspase-9 and -3 were detected by western blot. (B, C) Cleavage of caspase-9 and -3 in Bax/Bak wild-type and double knockout MEF cells after combination treatment. (D) Quantification of apoptotic cells by Annexin V and PI after 8 h treatment of combination at 50 μM. (E) Release of cytochrome c from Bax/Bak wild-type and double knockout MEF cells after combination treatment. These data represent three independent experiments.

    Article Snippet: Primary antibodies used were: Drp1 and Cytochrome c from BD Biosciences; β-actin and ATM from Sigma; Chk1, phospho-Chk2 (Thr68), Chk2, Mcl-1, Bax, Bak, Bid, Puma, Bik, Bcl-xL, Caspase-9 and cleaved Caspase-3 from Cell Signaling Technology; phospho-Chk1 (Ser317) from R & D systems; phospho-ATM (S1981) from Epitomics; phospho-Histone H2AX (Ser 139) and Noxa from Millipore; Complex IV subunit I from MitoSciences.

    Techniques: Double Knockout, Transformation Assay, Western Blot

    The combination of cisplatin and mdivi-1 preferentially upregulates Noxa and enhances subsequent mitochondrial apoptotic signaling (A) H1299 cells were treated with cisplatin alone, mdivi-1 alone, or the combination of cisplatin and mdivi-1 as indicated Western blot was then performed to detect the cleavage of caspase-9 and -3. (B) Cytochrome c release from mitochondria into cytosol. H1299 cells were treated with the combination of cisplatin and mdivi-1 at 50 μM with the presence of 20 μM caspase inhibitor Q-VD-OPH for the indicated time. The cytosol and heavy membrane fraction were then isolated using digitonin permeabilization followed by centrifugation. The amount of cytochrome c present in each fraction was detected by western blot. (C) The changes in the levels of pro-apoptotic and anti-apoptotic Bcl-2 family proteins. (D) The effect of cycloheximide on the levels of Noxa following the combination treatment. (E) H1299 cells were transfected with control or Noxa-specific siRNA for four days, and then treated with cisplatin and mdivi-1 as indicated. Noxa knockdown efficiency, mitochondrial release of cytochrome c, and the cleavage of caspase-9 and -3 were determined by western blot. These data represent three independent experiments.

    Journal: Oncotarget

    Article Title: Novel combination of mitochondrial division inhibitor 1 (mdivi-1) and platinum agents produces synergistic pro-apoptotic effect in drug resistant tumor cells

    doi:

    Figure Lengend Snippet: The combination of cisplatin and mdivi-1 preferentially upregulates Noxa and enhances subsequent mitochondrial apoptotic signaling (A) H1299 cells were treated with cisplatin alone, mdivi-1 alone, or the combination of cisplatin and mdivi-1 as indicated Western blot was then performed to detect the cleavage of caspase-9 and -3. (B) Cytochrome c release from mitochondria into cytosol. H1299 cells were treated with the combination of cisplatin and mdivi-1 at 50 μM with the presence of 20 μM caspase inhibitor Q-VD-OPH for the indicated time. The cytosol and heavy membrane fraction were then isolated using digitonin permeabilization followed by centrifugation. The amount of cytochrome c present in each fraction was detected by western blot. (C) The changes in the levels of pro-apoptotic and anti-apoptotic Bcl-2 family proteins. (D) The effect of cycloheximide on the levels of Noxa following the combination treatment. (E) H1299 cells were transfected with control or Noxa-specific siRNA for four days, and then treated with cisplatin and mdivi-1 as indicated. Noxa knockdown efficiency, mitochondrial release of cytochrome c, and the cleavage of caspase-9 and -3 were determined by western blot. These data represent three independent experiments.

    Article Snippet: Primary antibodies used were: Drp1 and Cytochrome c from BD Biosciences; β-actin and ATM from Sigma; Chk1, phospho-Chk2 (Thr68), Chk2, Mcl-1, Bax, Bak, Bid, Puma, Bik, Bcl-xL, Caspase-9 and cleaved Caspase-3 from Cell Signaling Technology; phospho-Chk1 (Ser317) from R & D systems; phospho-ATM (S1981) from Epitomics; phospho-Histone H2AX (Ser 139) and Noxa from Millipore; Complex IV subunit I from MitoSciences.

    Techniques: Western Blot, Isolation, Centrifugation, Transfection

    Apoptosis induced by Bcl-X L /Mcl-1 inhibition occurs in absence of activator BH3-only proteins. ( a ) Mcl-1-deficient MEFs were treated with control siRNAs or siRNAs targeting Puma, Bid, and Bim simultaneously. mRNA levels were measured 24 h after transfection. Data show means±S.D. of three independent experiments. ( b ) Mcl-1-deficient MEFs were transfected with control siRNAs or siRNAs targeting Puma, Bid and Bim. Forty-eight hour after transfection, cells were transfected with Bcl-X L or control siRNAs and cell death was measured 24 h later. Data show means±S.D. of three independent experiments. ( c ) MEFs from Bim/Puma-double-deficient or WT mice were transfected with Bcl-X L and Mcl-1 or control siRNAs. Cell death was measured 3 days after transfection. ( d ) MEFs from Bim/Puma-double-deficient mice were transfected with control or Bid-specific siRNA for 3 days. Then, cells were seeded and transfected with control, Bcl-X L -, Mcl-1- and Bid-specific siRNAs as indicated. Bid was quantified by immunoblotting 3 days after the second transfection. Results are representative of three independent experiments. ( e ) Cell death was measured in Bim/Puma-double-deficient MEFs treated as described in d . The mean percentage±S.D. of Annexin V-/propidium iodide-positive cells of three independent experiments is given. ( f ) Upper panel: representative histogram of Bim/Puma-double-deficient MEFs treated as described in d indicating cells undergoing apoptosis as demonstrated by an Annexin V-positive/propidium iodide-negative cell population. Lower panel: quantification of caspase-3 activation by immunoblotting. Results are representative of three independent experiments. ( g ) Bid/Bim/Puma-triple-deficient MEFs were transfected with Bcl-X L - and Mcl-1-specific siRNAs or control siRNAs. Cell death was assessed after 48 h. For Bcl-X L and Mcl-1 two different siRNA sequences (siRNA a or b) were used in different combinations. Inset shows result of knockout of Bid using CRISPR/CAS9 on Bim/Puma DKO MEF to generate triple KO cells. ( h ) Primary human fibroblasts were treated simultaneously with Bcl-X L -, Mcl-1- and Bak-specific siRNAs. Three days after transfection mitochondria were prepared. Mitochondria were treated with recombinant Bax (1000 nM) to stimulate cytochrome c (Cyt- c ) release. Cyt- c release was assessed by immunoblotting in supernatant (SN) and mitochondrial (M) fractions. Tom40 (translocase of the outer mitochondrial membrane protein 40) served as a control for mitochondria preparation. ( i ) Mitochondria of Bax/Bak-double-deficient MEFs were treated with recombinant Bax. Cyt- c release was measured with 1000 nM Bax. A representative blot of two experiments is shown. * P ≤0.05

    Journal: Cell Death & Disease

    Article Title: In non-transformed cells Bak activates upon loss of anti-apoptotic Bcl-XL and Mcl-1 but in the absence of active BH3-only proteins

    doi: 10.1038/cddis.2015.341

    Figure Lengend Snippet: Apoptosis induced by Bcl-X L /Mcl-1 inhibition occurs in absence of activator BH3-only proteins. ( a ) Mcl-1-deficient MEFs were treated with control siRNAs or siRNAs targeting Puma, Bid, and Bim simultaneously. mRNA levels were measured 24 h after transfection. Data show means±S.D. of three independent experiments. ( b ) Mcl-1-deficient MEFs were transfected with control siRNAs or siRNAs targeting Puma, Bid and Bim. Forty-eight hour after transfection, cells were transfected with Bcl-X L or control siRNAs and cell death was measured 24 h later. Data show means±S.D. of three independent experiments. ( c ) MEFs from Bim/Puma-double-deficient or WT mice were transfected with Bcl-X L and Mcl-1 or control siRNAs. Cell death was measured 3 days after transfection. ( d ) MEFs from Bim/Puma-double-deficient mice were transfected with control or Bid-specific siRNA for 3 days. Then, cells were seeded and transfected with control, Bcl-X L -, Mcl-1- and Bid-specific siRNAs as indicated. Bid was quantified by immunoblotting 3 days after the second transfection. Results are representative of three independent experiments. ( e ) Cell death was measured in Bim/Puma-double-deficient MEFs treated as described in d . The mean percentage±S.D. of Annexin V-/propidium iodide-positive cells of three independent experiments is given. ( f ) Upper panel: representative histogram of Bim/Puma-double-deficient MEFs treated as described in d indicating cells undergoing apoptosis as demonstrated by an Annexin V-positive/propidium iodide-negative cell population. Lower panel: quantification of caspase-3 activation by immunoblotting. Results are representative of three independent experiments. ( g ) Bid/Bim/Puma-triple-deficient MEFs were transfected with Bcl-X L - and Mcl-1-specific siRNAs or control siRNAs. Cell death was assessed after 48 h. For Bcl-X L and Mcl-1 two different siRNA sequences (siRNA a or b) were used in different combinations. Inset shows result of knockout of Bid using CRISPR/CAS9 on Bim/Puma DKO MEF to generate triple KO cells. ( h ) Primary human fibroblasts were treated simultaneously with Bcl-X L -, Mcl-1- and Bak-specific siRNAs. Three days after transfection mitochondria were prepared. Mitochondria were treated with recombinant Bax (1000 nM) to stimulate cytochrome c (Cyt- c ) release. Cyt- c release was assessed by immunoblotting in supernatant (SN) and mitochondrial (M) fractions. Tom40 (translocase of the outer mitochondrial membrane protein 40) served as a control for mitochondria preparation. ( i ) Mitochondria of Bax/Bak-double-deficient MEFs were treated with recombinant Bax. Cyt- c release was measured with 1000 nM Bax. A representative blot of two experiments is shown. * P ≤0.05

    Article Snippet: Anti-Mcl-1 (22) antibody was from BD Bioscience (Heidelberg, Germany), anti-Bax (2D2) was from Santa Cruz Biotechnology (Heidelberg, Germany) and anti-cytochrome c (clone 7H8.2C12) was purchased from BD Biosciences (Heidelberg, Germany).

    Techniques: Inhibition, Transfection, Mouse Assay, Activation Assay, Knock-Out, CRISPR, Recombinant

    PERK deficiency protects cells from ROS-induced mitochondrial dysfunction. ( a ) Comparison of cardiolipin oxidation following phox-ER stress in PERK +/+ and PERK −/− MEFs. Cells were left untreated or treated with phox-ER stress. At the indicated time points, cells were incubated with 10 nM NAO and the percentage of cells with reduced cardiolipin binding (NAO−) was determined by flow cytometry. Left: histograms showing results for a representative experiment; right: graph represents the mean±S.D. of three independent experiments performed in duplicate. ( b ) Release of cytochrome c (cyt c ) following phox-ER stress in PERK +/+ and PERK −/− MEFs. Cells were left untreated (Ctr) or phox treated. At the indicated time points, subcellular fractions were made for western blot analysis of cytochrome c release in the cytosol. A representative western blot from three independent experiments is shown. Left and right parts are from the same membrane. ( c ) Comparison of cardiolipin oxidation following phox-ER stress in CHOP +/+ and CHOP −/− MEFs. Cells were left untreated (Ctr) or treated with phox-ER stress (1.62 J/cm 2 ). At the indicated time points, cells were incubated with 10 nM NAO and the percentage of cells with reduced cardiolipin binding (NAO) was determined by flow cytometry. Graph represents the mean±S.D. of three independent experiments performed in duplicate. ( d ) Dissipation of mitochondrial membrane potential (Δψ m ) following phox-ER stress in PERK +/+ and PERK −/− MEFs. Cells were left untreated (Ctr) or phox treated at the indicated irradiation dose. After 6 h, cells were incubated with 2 nM DiOC6(3) and the percentage of cells with reduced membrane potential (DiOC6(3)−) was determined by flow cytometry. Left: histograms showing results for a representative experiment. Right: graph represents the mean±S.D. of three independent experiments. (*) indicates P

    Journal: Cell Death and Differentiation

    Article Title: PERK is required at the ER-mitochondrial contact sites to convey apoptosis after ROS-based ER stress

    doi: 10.1038/cdd.2012.74

    Figure Lengend Snippet: PERK deficiency protects cells from ROS-induced mitochondrial dysfunction. ( a ) Comparison of cardiolipin oxidation following phox-ER stress in PERK +/+ and PERK −/− MEFs. Cells were left untreated or treated with phox-ER stress. At the indicated time points, cells were incubated with 10 nM NAO and the percentage of cells with reduced cardiolipin binding (NAO−) was determined by flow cytometry. Left: histograms showing results for a representative experiment; right: graph represents the mean±S.D. of three independent experiments performed in duplicate. ( b ) Release of cytochrome c (cyt c ) following phox-ER stress in PERK +/+ and PERK −/− MEFs. Cells were left untreated (Ctr) or phox treated. At the indicated time points, subcellular fractions were made for western blot analysis of cytochrome c release in the cytosol. A representative western blot from three independent experiments is shown. Left and right parts are from the same membrane. ( c ) Comparison of cardiolipin oxidation following phox-ER stress in CHOP +/+ and CHOP −/− MEFs. Cells were left untreated (Ctr) or treated with phox-ER stress (1.62 J/cm 2 ). At the indicated time points, cells were incubated with 10 nM NAO and the percentage of cells with reduced cardiolipin binding (NAO) was determined by flow cytometry. Graph represents the mean±S.D. of three independent experiments performed in duplicate. ( d ) Dissipation of mitochondrial membrane potential (Δψ m ) following phox-ER stress in PERK +/+ and PERK −/− MEFs. Cells were left untreated (Ctr) or phox treated at the indicated irradiation dose. After 6 h, cells were incubated with 2 nM DiOC6(3) and the percentage of cells with reduced membrane potential (DiOC6(3)−) was determined by flow cytometry. Left: histograms showing results for a representative experiment. Right: graph represents the mean±S.D. of three independent experiments. (*) indicates P

    Article Snippet: Anti-cytochrome c antibodies were from BD Pharmingen (San Jose, CA, USA), Antibodies against TOM20, Calreticulin (CRT), MFN2 and VDAC-2 were from Abcam (Cambridge, UK) and anti-IP3 R3 antibody was from BD Biosciences (Franklin Lakes, NJ, USA).

    Techniques: Incubation, Binding Assay, Flow Cytometry, Cytometry, Western Blot, Irradiation

    PERK has a dual role in ER stress signaling. PERK regulates photo-oxidative (phox)–ER stress induced apoptosis via two distinct mechanisms. On the one hand, canonical signaling via the PERK-eIF2 α -ATF4 branch of the UPR induces the pro-apoptotic transcription factor CHOP, which in turn mediates mitochondrial apoptosis through the upregulation of BH3 only proteins, like Bim, leading to Bax activation. On the other hand, PERK is required at the MAMs for tethering the ER to mitochondria, thereby promoting the rapid transfer of ROS signals, likely under the form of lipid hydroperoxides, from the ER to the mitochondria. As a result, the mitochondrial phospholipid cardiolipin is oxidized (and possibly externalized to the outer mitochondrial membrane) and cytochrome c is released from the pool of mitochondria in close contact with the ER (where ROS are formed). Sustained cardiolipin oxidation may contribute to the insertion of Bax into the outer mitochondrial membrane inducing full blown mitochondrial apoptosis

    Journal: Cell Death and Differentiation

    Article Title: PERK is required at the ER-mitochondrial contact sites to convey apoptosis after ROS-based ER stress

    doi: 10.1038/cdd.2012.74

    Figure Lengend Snippet: PERK has a dual role in ER stress signaling. PERK regulates photo-oxidative (phox)–ER stress induced apoptosis via two distinct mechanisms. On the one hand, canonical signaling via the PERK-eIF2 α -ATF4 branch of the UPR induces the pro-apoptotic transcription factor CHOP, which in turn mediates mitochondrial apoptosis through the upregulation of BH3 only proteins, like Bim, leading to Bax activation. On the other hand, PERK is required at the MAMs for tethering the ER to mitochondria, thereby promoting the rapid transfer of ROS signals, likely under the form of lipid hydroperoxides, from the ER to the mitochondria. As a result, the mitochondrial phospholipid cardiolipin is oxidized (and possibly externalized to the outer mitochondrial membrane) and cytochrome c is released from the pool of mitochondria in close contact with the ER (where ROS are formed). Sustained cardiolipin oxidation may contribute to the insertion of Bax into the outer mitochondrial membrane inducing full blown mitochondrial apoptosis

    Article Snippet: Anti-cytochrome c antibodies were from BD Pharmingen (San Jose, CA, USA), Antibodies against TOM20, Calreticulin (CRT), MFN2 and VDAC-2 were from Abcam (Cambridge, UK) and anti-IP3 R3 antibody was from BD Biosciences (Franklin Lakes, NJ, USA).

    Techniques: Activation Assay, Micro-arrays for Mass Spectrometry

    Inhibition of p210 Bcr-Abl leads to increased JNK activity. (A) K562 cells were treated with imatinib with 1µM for 24 hr and the levels of phosphorylated p38 MAPK, p38MAPK, phosphorylated SAPK/JNK, SAPK/JNK phosphorylated p42/44 MAPK, p42/44 MAPK was assessed. Beta actin was used as a loading control. (B) K562 cells were transfected with MOCK, RhoAN19 or RhoAL63 vectors followed by treatment with/without 1µM imatinib for 24 hr. The levels of phosphorylated p38 MAPK, p38MAPK and phosphorylated SAPK/JNK, SAPK/JNK was assessed by western blotting. (C) K562 cells were treated with imatinib, C3 exozyme or both and the activity of SAPK/JNK pathway was assessed by looking into the phosphorylation of c-jun. (D) K562 cells were treated with imatinib, SP600125 or both and the level of cytochrome c released from the mitochondria was analysed in the mitochondria free cytosolic fraction. Tim23 was used to estimate mitochondrial contamination in the mitochondria free cytosolic preparations while beta actin was used as a loading control. (E) K562 cells were treated with the indicated concentrations of imatinib and SP600125 either singly or in combination for 24hrs. The percentage metabolic activity was assessed by MTT assay. Data shows the mean percent metabolic activity for individual treatments in three separate experiments. (F) K562 cells were treated with 1µM imatinib, 20µM SP600125 or both and apoptosis was measured by staining the cells with Annexin V and PI. Graph shows the change in Annexin V stained and Annexin V + PI stained population compared to untreated control cells (n=3, *p

    Journal: PLoS ONE

    Article Title: Increased Cytoplasmic Localization of p27kip1 and Its Modulation of RhoA Activity during Progression of Chronic Myeloid Leukemia

    doi: 10.1371/journal.pone.0076527

    Figure Lengend Snippet: Inhibition of p210 Bcr-Abl leads to increased JNK activity. (A) K562 cells were treated with imatinib with 1µM for 24 hr and the levels of phosphorylated p38 MAPK, p38MAPK, phosphorylated SAPK/JNK, SAPK/JNK phosphorylated p42/44 MAPK, p42/44 MAPK was assessed. Beta actin was used as a loading control. (B) K562 cells were transfected with MOCK, RhoAN19 or RhoAL63 vectors followed by treatment with/without 1µM imatinib for 24 hr. The levels of phosphorylated p38 MAPK, p38MAPK and phosphorylated SAPK/JNK, SAPK/JNK was assessed by western blotting. (C) K562 cells were treated with imatinib, C3 exozyme or both and the activity of SAPK/JNK pathway was assessed by looking into the phosphorylation of c-jun. (D) K562 cells were treated with imatinib, SP600125 or both and the level of cytochrome c released from the mitochondria was analysed in the mitochondria free cytosolic fraction. Tim23 was used to estimate mitochondrial contamination in the mitochondria free cytosolic preparations while beta actin was used as a loading control. (E) K562 cells were treated with the indicated concentrations of imatinib and SP600125 either singly or in combination for 24hrs. The percentage metabolic activity was assessed by MTT assay. Data shows the mean percent metabolic activity for individual treatments in three separate experiments. (F) K562 cells were treated with 1µM imatinib, 20µM SP600125 or both and apoptosis was measured by staining the cells with Annexin V and PI. Graph shows the change in Annexin V stained and Annexin V + PI stained population compared to untreated control cells (n=3, *p

    Article Snippet: Antibody against cytochrome c was obtained from BD Biosciences (CA, USA).

    Techniques: Inhibition, Activity Assay, Transfection, Western Blot, MTT Assay, Staining

    Expression levels of apoptotic proteins after SIRT1 silencing following ICH.  (A)  Representative western blots of AIF, cytochrome c, caspase-3, and cleaved caspase-3. Relative protein band density values were calculated as the ratio of the protein of interest to that of GAPDH. Quantification of  (A)  is shown in  (B) . Error bars represent mean ± SEM. ( ∗ P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: SIRT1/PGC-1α Signaling Promotes Mitochondrial Functional Recovery and Reduces Apoptosis after Intracerebral Hemorrhage in Rats

    doi: 10.3389/fnmol.2017.00443

    Figure Lengend Snippet: Expression levels of apoptotic proteins after SIRT1 silencing following ICH. (A) Representative western blots of AIF, cytochrome c, caspase-3, and cleaved caspase-3. Relative protein band density values were calculated as the ratio of the protein of interest to that of GAPDH. Quantification of (A) is shown in (B) . Error bars represent mean ± SEM. ( ∗ P

    Article Snippet: Antibodies used were as follows: SIRT1 (1:500, Cell Signaling Technologies, Danvers, MA), PGC-1α (1:1,000, Abcam, Cambridge, MA, United States), AMP-activated protein kinase (AMPK) (1:1,000, Proteintech), phosphorylated AMP-activated protein kinase (pAMPK) (1:500, Bioworld), acetylated-lysine (1:500, Bioworld), lamin B1 (Cell Signaling Technologies), GAPDH (1:2000, Bioworld), ATP synthase β (ATPβ) (1:400, Bioworld), cytochrome c oxidase subunit I (1:400, Bioworld), NADH dehydrogenase (ubiquinone) 1 beta subcomplex subunit 8 (NDUFB8) (1:400, Bioworld), cytochrome c (1:1,000, Abcam), AIF (1:1,000, Santa Cruz, Dallas, TX), cleaved caspase-3 (cleaved c3) (1:1,000, Cell Signaling Technologies, Danvers, MA), and caspase-3 (c3) (1:500, Santa Cruz).

    Techniques: Expressing, Western Blot

    Expression levels of apoptotic proteins after ICH injury following SIRT1 activation in rats. Representative western blots of AIF, cytochrome c, caspase-3 (c3), and cleaved caspase-3 (cleaved c3)  (A,B) . Relative protein band density values were calculated as the ratio of the protein of interest to that of GAPDH. Quantification of  (A,B)  is shown in  (C,D) , respectively. Error bars represent mean ± SEM.  (C)  ( ∗ P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: SIRT1/PGC-1α Signaling Promotes Mitochondrial Functional Recovery and Reduces Apoptosis after Intracerebral Hemorrhage in Rats

    doi: 10.3389/fnmol.2017.00443

    Figure Lengend Snippet: Expression levels of apoptotic proteins after ICH injury following SIRT1 activation in rats. Representative western blots of AIF, cytochrome c, caspase-3 (c3), and cleaved caspase-3 (cleaved c3) (A,B) . Relative protein band density values were calculated as the ratio of the protein of interest to that of GAPDH. Quantification of (A,B) is shown in (C,D) , respectively. Error bars represent mean ± SEM. (C) ( ∗ P

    Article Snippet: Antibodies used were as follows: SIRT1 (1:500, Cell Signaling Technologies, Danvers, MA), PGC-1α (1:1,000, Abcam, Cambridge, MA, United States), AMP-activated protein kinase (AMPK) (1:1,000, Proteintech), phosphorylated AMP-activated protein kinase (pAMPK) (1:500, Bioworld), acetylated-lysine (1:500, Bioworld), lamin B1 (Cell Signaling Technologies), GAPDH (1:2000, Bioworld), ATP synthase β (ATPβ) (1:400, Bioworld), cytochrome c oxidase subunit I (1:400, Bioworld), NADH dehydrogenase (ubiquinone) 1 beta subcomplex subunit 8 (NDUFB8) (1:400, Bioworld), cytochrome c (1:1,000, Abcam), AIF (1:1,000, Santa Cruz, Dallas, TX), cleaved caspase-3 (cleaved c3) (1:1,000, Cell Signaling Technologies, Danvers, MA), and caspase-3 (c3) (1:500, Santa Cruz).

    Techniques: Expressing, Activation Assay, Western Blot

    The effect of SSB consumption on calpain and cytochrome c expression. ( A ) Calpain and ( B ) cytochrome c levels. Data are displayed as mean ± SEM ( n = 8).

    Journal: Antioxidants

    Article Title: The Impact of Sugar-Sweetened Beverage Consumption on the Liver: A Proteomics-Based Analysis

    doi: 10.3390/antiox9070569

    Figure Lengend Snippet: The effect of SSB consumption on calpain and cytochrome c expression. ( A ) Calpain and ( B ) cytochrome c levels. Data are displayed as mean ± SEM ( n = 8).

    Article Snippet: Protein expression was detected with anti-DRP1 (DNM1L, ab56788), anti-MFN2 (ab50843), anti-PGC1α (ab106814), anti-IP3R (Cell Signaling Technology® , Danvers, MA, USA, 8568), anti-porin (VDAC1, ab15895), anti-IRE1 (ab48187) anti-PERK (ab79483), anti-ATF6 (Novus Biologicals, Centennial, CO, USA, 70B1413.1), anti-CHOP (Cell Signaling Technology® , 2895), anti-calpain-1 large subunit (Cell Signaling Technology® , 2556) and anti-cytochrome c (Cell Signaling Technology®, 6H2.B4).

    Techniques: Expressing

    Bax translocation, cytochrome c release, and cell death following PV infection depend on JNK activation. (A) Inhibition of JNK activity during PV infection in IMR5 cells treated with SP600125 (25 μM). Cells were incubated with the JNK inhibitor for 2 h before PV infection, and the inhibitor concentration was maintained during the adsorption period and throughout PV infection. Levels of phospho (Thr183/Tyr185)-JNK and phospho (Ser63)-c-Jun in whole-cell lysates were determined at 30 min p.i. by Western blotting. Blots were subsequently stripped and reprobed with antibodies recognizing all JNK and c-Jun forms to confirm equal protein loading. (B) IMR5 cells were uninfected or were infected with PV in the presence or absence of SP600125 (25 μM). Whole-cell lysates (8 h p.i.) were subjected to Western blot analysis with anti-Bax and anti-cytochrome c (Cyt c) antibodies. Actin was used as a control for protein loading. (C) Inhibition of Bax translocation from the cytosol to mitochondria by the JNK inhibitor SP600125. IMR5 cells were uninfected or were infected with PV in the presence or the absence of SP600125 (25 μM). Eight hours p.i., the cytosolic and heavy membrane fractions were assayed for Bax by Western blotting. Actin and Cox IV were used as controls for protein loading of cytosolic and heavy membrane fractions, respectively. Protein levels of heavy membrane and cytosolic fractions were determined by densitometry and plotted as ratios relative to the levels of Cox IV and actin, respectively. (D) Cytochrome c release is reduced by JNK inhibitor. IMR5 cells were uninfected or were infected with PV in the presence or absence of SP600125 (25 μM). Cytosolic extract proteins were analyzed at 14 h p.i. by immunoblotting with anti-cytochrome c (Cyt c) antibody. Actin was used as a control for protein loading. Protein levels were determined by densitometry and plotted as ratios relative to the levels of actin. (E) Inhibition of PV-induced cytopathic effect by the JNK inhibitor SP600125. Cells were uninfected or were infected with PV in the presence or absence of SP600125 (25 μM) and visualized by light microscopy at the indicated times p.i. Magnification, ×350. (F) The JNK inhibitor SP600125 does not affect PV growth but affects PV release. IMR5 cells were infected with PV in the presence or absence of SP600125 (25 μM). Total virus yield (extracellular and intracellular) was determined by TCID 50 assay at the indicated times after three cycles of freezing and thawing to release intracellular viruses. The titers of extracellular virus were determined from the supernatant of PV-infected cells at the indicated times after the removal of detached cells by centrifugation. Each point represents the mean virus titer for two independent experiments. Standard errors of the means are indicated. *, P

    Journal: Journal of Virology

    Article Title: Poliovirus Induces Bax-Dependent Cell Death Mediated by c-Jun NH2-Terminal Kinase ▿

    doi: 10.1128/JVI.02690-06

    Figure Lengend Snippet: Bax translocation, cytochrome c release, and cell death following PV infection depend on JNK activation. (A) Inhibition of JNK activity during PV infection in IMR5 cells treated with SP600125 (25 μM). Cells were incubated with the JNK inhibitor for 2 h before PV infection, and the inhibitor concentration was maintained during the adsorption period and throughout PV infection. Levels of phospho (Thr183/Tyr185)-JNK and phospho (Ser63)-c-Jun in whole-cell lysates were determined at 30 min p.i. by Western blotting. Blots were subsequently stripped and reprobed with antibodies recognizing all JNK and c-Jun forms to confirm equal protein loading. (B) IMR5 cells were uninfected or were infected with PV in the presence or absence of SP600125 (25 μM). Whole-cell lysates (8 h p.i.) were subjected to Western blot analysis with anti-Bax and anti-cytochrome c (Cyt c) antibodies. Actin was used as a control for protein loading. (C) Inhibition of Bax translocation from the cytosol to mitochondria by the JNK inhibitor SP600125. IMR5 cells were uninfected or were infected with PV in the presence or the absence of SP600125 (25 μM). Eight hours p.i., the cytosolic and heavy membrane fractions were assayed for Bax by Western blotting. Actin and Cox IV were used as controls for protein loading of cytosolic and heavy membrane fractions, respectively. Protein levels of heavy membrane and cytosolic fractions were determined by densitometry and plotted as ratios relative to the levels of Cox IV and actin, respectively. (D) Cytochrome c release is reduced by JNK inhibitor. IMR5 cells were uninfected or were infected with PV in the presence or absence of SP600125 (25 μM). Cytosolic extract proteins were analyzed at 14 h p.i. by immunoblotting with anti-cytochrome c (Cyt c) antibody. Actin was used as a control for protein loading. Protein levels were determined by densitometry and plotted as ratios relative to the levels of actin. (E) Inhibition of PV-induced cytopathic effect by the JNK inhibitor SP600125. Cells were uninfected or were infected with PV in the presence or absence of SP600125 (25 μM) and visualized by light microscopy at the indicated times p.i. Magnification, ×350. (F) The JNK inhibitor SP600125 does not affect PV growth but affects PV release. IMR5 cells were infected with PV in the presence or absence of SP600125 (25 μM). Total virus yield (extracellular and intracellular) was determined by TCID 50 assay at the indicated times after three cycles of freezing and thawing to release intracellular viruses. The titers of extracellular virus were determined from the supernatant of PV-infected cells at the indicated times after the removal of detached cells by centrifugation. Each point represents the mean virus titer for two independent experiments. Standard errors of the means are indicated. *, P

    Article Snippet: Mouse anti-cytochrome c (7H8.2C12) and anti-JNK1/2 (G151-666) antibodies were obtained from BD Pharmingen.

    Techniques: Translocation Assay, Infection, Activation Assay, Inhibition, Activity Assay, Incubation, Concentration Assay, Adsorption, Western Blot, Light Microscopy, Centrifugation

    PV-mediated cytochrome c release in neuronal cells is Bax dependent. (A) Translocation of the proapoptotic protein Bax to mitochondria in PV-infected IMR5 cells. At the indicated time p.i., equal amounts of cytosolic and mitochondrial proteins were assayed for Bax by Western blotting. Mock-infected and STS-treated (8 h posttreatment) IMR5 cells were used as negative and positive controls, respectively. Cox IV and actin were used as protein loading controls for heavy membrane and cytosolic fractions, respectively. Protein levels of heavy membrane and cytosolic fractions were determined by densitometry and plotted as ratios relative to the levels of Cox IV and actin, respectively. (B) Time course of PV-induced Bax conformational change in IMR5 cells. (Top) Mock-infected and PV-infected IMR5 cells were lysed in immunoprecipitation buffer. Conformationally active Bax protein was immunoprecipitated (IP) with anti-Bax 6A7 antibody and the precipitates were immunoblotted with anti-Bax antibody. The asterisk indicates immunoglobulin light chains. (Bottom) Whole-cell lysates that were not incubated with antibody were similarly tested for Bax by immunoblotting to check for equal amounts of Bax protein in samples prior to immunoprecipitation. Actin was used as protein loading controls. (C) PV-induced Bax activation. Mock-infected and PV-infected (6 h p.i.) IMR5 cells were analyzed by immunofluorescence with an antibody specific for the N terminus of Bax (NT antibody) to detect Bax conformational change. (D) Inhibition of cytochrome c release after vMIA expression in PV-infected IMR5 cells. (Top) Cells were transfected with plasmids encoding Myc-tagged vMIA, were transfected with empty vector (pcDNA3.1), or were left untreated. vMIA protein was detected in whole-cell lysates by Western blotting analysis with anti-Myc antibody. Actin was used as a protein loading control. The asterisk indicates a probable nonspecific protein band. (Bottom) Cytochrome c (Cyt c) release 24 h after transfection was analyzed in cytosolic fractions of mock-infected and PV-infected cells (8 h p.i.) by immunoblotting. Protein levels were determined by densitometry and plotted as ratios relative to the levels of actin.

    Journal: Journal of Virology

    Article Title: Poliovirus Induces Bax-Dependent Cell Death Mediated by c-Jun NH2-Terminal Kinase ▿

    doi: 10.1128/JVI.02690-06

    Figure Lengend Snippet: PV-mediated cytochrome c release in neuronal cells is Bax dependent. (A) Translocation of the proapoptotic protein Bax to mitochondria in PV-infected IMR5 cells. At the indicated time p.i., equal amounts of cytosolic and mitochondrial proteins were assayed for Bax by Western blotting. Mock-infected and STS-treated (8 h posttreatment) IMR5 cells were used as negative and positive controls, respectively. Cox IV and actin were used as protein loading controls for heavy membrane and cytosolic fractions, respectively. Protein levels of heavy membrane and cytosolic fractions were determined by densitometry and plotted as ratios relative to the levels of Cox IV and actin, respectively. (B) Time course of PV-induced Bax conformational change in IMR5 cells. (Top) Mock-infected and PV-infected IMR5 cells were lysed in immunoprecipitation buffer. Conformationally active Bax protein was immunoprecipitated (IP) with anti-Bax 6A7 antibody and the precipitates were immunoblotted with anti-Bax antibody. The asterisk indicates immunoglobulin light chains. (Bottom) Whole-cell lysates that were not incubated with antibody were similarly tested for Bax by immunoblotting to check for equal amounts of Bax protein in samples prior to immunoprecipitation. Actin was used as protein loading controls. (C) PV-induced Bax activation. Mock-infected and PV-infected (6 h p.i.) IMR5 cells were analyzed by immunofluorescence with an antibody specific for the N terminus of Bax (NT antibody) to detect Bax conformational change. (D) Inhibition of cytochrome c release after vMIA expression in PV-infected IMR5 cells. (Top) Cells were transfected with plasmids encoding Myc-tagged vMIA, were transfected with empty vector (pcDNA3.1), or were left untreated. vMIA protein was detected in whole-cell lysates by Western blotting analysis with anti-Myc antibody. Actin was used as a protein loading control. The asterisk indicates a probable nonspecific protein band. (Bottom) Cytochrome c (Cyt c) release 24 h after transfection was analyzed in cytosolic fractions of mock-infected and PV-infected cells (8 h p.i.) by immunoblotting. Protein levels were determined by densitometry and plotted as ratios relative to the levels of actin.

    Article Snippet: Mouse anti-cytochrome c (7H8.2C12) and anti-JNK1/2 (G151-666) antibodies were obtained from BD Pharmingen.

    Techniques: Translocation Assay, Infection, Western Blot, Immunoprecipitation, Incubation, Activation Assay, Immunofluorescence, Inhibition, Expressing, Transfection, Plasmid Preparation

    PV-mediated MOMP in neuronal cells is Bid and Bim independent. (A) Time course of caspase-8 and Bid processing in PV-infected IMR5 cells. (Top) At the indicated times p.i., whole-cell extracts were subjected to immunoblot analysis with anti-caspase-8 (Pro-C8) and anti-Bid antibodies. Actin was used as a control for protein loading. (Bottom) Caspase-8 activation in PV-infected IMR5 cells. Caspase-8 activation was determined in mock- and PV-infected IMR5 cells (14 h p.i.) by flow cytometry using a fluorescein-labeled inhibitor (FAM-LETD-fmk) that binds specifically to active caspase-8, as described in Materials and Methods. Histograms representative of two independent experiments are shown. The percentages of cells positive for activated caspase-8 are indicated. (B) The broad-spectrum caspase inhibitor z-VAD-fmk does not inhibit cytochrome c release during PV infection. Mock- and PV-infected IMR5 cells were left untreated or treated with 100 μM z-VAD-fmk for 2 h before PV infection, and the inhibitor concentration was maintained during the adsorption period and throughout PV infection. Cytochrome c (Cyt c) was assayed in cytosolic extracts by Western blot analysis (14 h p.i.). Actin was used as a control for protein loading. (C) No inhibition of cytochrome c release after knockdown of Bim expression in PV-infected cells. (Top) IMR5 cells were transfected with Bim siRNA or nontargeted control siRNA. Bim protein was then assayed by immunoblotting with extracts from nontargeted control siRNA-transfected and Bim siRNA-transfected cells. Actin was used as a protein loading control. (Bottom) Cells were uninfected or were infected with PV 72 h after transfection, and cytochrome c (Cyt c) release was analyzed in cytosolic fractions by Western blotting 8 h p.i. Actin was used as a protein loading control. Protein levels were determined by densitometry and plotted as ratios relative to the levels of actin.

    Journal: Journal of Virology

    Article Title: Poliovirus Induces Bax-Dependent Cell Death Mediated by c-Jun NH2-Terminal Kinase ▿

    doi: 10.1128/JVI.02690-06

    Figure Lengend Snippet: PV-mediated MOMP in neuronal cells is Bid and Bim independent. (A) Time course of caspase-8 and Bid processing in PV-infected IMR5 cells. (Top) At the indicated times p.i., whole-cell extracts were subjected to immunoblot analysis with anti-caspase-8 (Pro-C8) and anti-Bid antibodies. Actin was used as a control for protein loading. (Bottom) Caspase-8 activation in PV-infected IMR5 cells. Caspase-8 activation was determined in mock- and PV-infected IMR5 cells (14 h p.i.) by flow cytometry using a fluorescein-labeled inhibitor (FAM-LETD-fmk) that binds specifically to active caspase-8, as described in Materials and Methods. Histograms representative of two independent experiments are shown. The percentages of cells positive for activated caspase-8 are indicated. (B) The broad-spectrum caspase inhibitor z-VAD-fmk does not inhibit cytochrome c release during PV infection. Mock- and PV-infected IMR5 cells were left untreated or treated with 100 μM z-VAD-fmk for 2 h before PV infection, and the inhibitor concentration was maintained during the adsorption period and throughout PV infection. Cytochrome c (Cyt c) was assayed in cytosolic extracts by Western blot analysis (14 h p.i.). Actin was used as a control for protein loading. (C) No inhibition of cytochrome c release after knockdown of Bim expression in PV-infected cells. (Top) IMR5 cells were transfected with Bim siRNA or nontargeted control siRNA. Bim protein was then assayed by immunoblotting with extracts from nontargeted control siRNA-transfected and Bim siRNA-transfected cells. Actin was used as a protein loading control. (Bottom) Cells were uninfected or were infected with PV 72 h after transfection, and cytochrome c (Cyt c) release was analyzed in cytosolic fractions by Western blotting 8 h p.i. Actin was used as a protein loading control. Protein levels were determined by densitometry and plotted as ratios relative to the levels of actin.

    Article Snippet: Mouse anti-cytochrome c (7H8.2C12) and anti-JNK1/2 (G151-666) antibodies were obtained from BD Pharmingen.

    Techniques: Infection, Activation Assay, Flow Cytometry, Cytometry, Labeling, Concentration Assay, Adsorption, Western Blot, Inhibition, Expressing, Transfection

    Hydrodynamic radius ( a ) and electric dipole moment ( b ) of cytochrome-c in water/ethylene-glycol mixtures as function of weight fraction of cosolvent. The error bars are the result of a statistical analysis connected to the best fit procedure employed to analyze the experimental data. Level of confidence 95%. Dotted lines for visual aim.

    Journal: Biophysical Journal

    Article Title: Dielectric Behavior of Lysozyme and Ferricytochrome-c in Water/Ethylene-Glycol Solutions

    doi:

    Figure Lengend Snippet: Hydrodynamic radius ( a ) and electric dipole moment ( b ) of cytochrome-c in water/ethylene-glycol mixtures as function of weight fraction of cosolvent. The error bars are the result of a statistical analysis connected to the best fit procedure employed to analyze the experimental data. Level of confidence 95%. Dotted lines for visual aim.

    Article Snippet: Chicken egg-white lysozyme and horse heart cytochrome-c were obtained from Sigma (St. Louis, MS) and used without ulterior purification.

    Techniques:

    Caspase independent cell death MCF7 cells were exposed to air, hypoxia (Hx) or hypoxia with Baf1A for 36 hrs and the cells fractionated into soluble (cytoplasmic) and heavy membrane (mitochondrial) fractions. Cytoplasmic levels of cytochrome c is shown in (A). Fractional purity was determined by the mitochondrial marker VDAC. In (B) caspase 3 enzymatic activity was determined in MCF7 cells exposed to air (48 hrs) or to hypoxia (Hx) in the presence and absence of Baf1A for times indicated. The level of cleaved ICAD, a target of caspase 3, was determined in hypoxic MCF7 cells exposed to increasing concentrations of Baf1A (C). As a positive control a parallel plate was treated with 1 μM staurosporine, a potent inducer of caspase dependent apoptosis. In (D), proteolysis of the calpain target, α-fodrin, was determined in MCF7 cell exposed to hypoxia and hypoxia-Baf1A for indicated times. Data are means ± SEM. All results are representative of at least 3 experiments.

    Journal: Oncotarget

    Article Title: Inhibition of the vacuolar ATPase induces Bnip3-dependent death of cancer cells and a reduction in tumor burden and metastasis

    doi:

    Figure Lengend Snippet: Caspase independent cell death MCF7 cells were exposed to air, hypoxia (Hx) or hypoxia with Baf1A for 36 hrs and the cells fractionated into soluble (cytoplasmic) and heavy membrane (mitochondrial) fractions. Cytoplasmic levels of cytochrome c is shown in (A). Fractional purity was determined by the mitochondrial marker VDAC. In (B) caspase 3 enzymatic activity was determined in MCF7 cells exposed to air (48 hrs) or to hypoxia (Hx) in the presence and absence of Baf1A for times indicated. The level of cleaved ICAD, a target of caspase 3, was determined in hypoxic MCF7 cells exposed to increasing concentrations of Baf1A (C). As a positive control a parallel plate was treated with 1 μM staurosporine, a potent inducer of caspase dependent apoptosis. In (D), proteolysis of the calpain target, α-fodrin, was determined in MCF7 cell exposed to hypoxia and hypoxia-Baf1A for indicated times. Data are means ± SEM. All results are representative of at least 3 experiments.

    Article Snippet: Western blot analysis was performed using antibodies to Bnip3 (Abcam), V-ATPase (EMD Millipore), PUMA, NOXA, Bim, Mcl-xl, Bcl-2, Bak (Cell Signaling Technologies), cytochrome c (Pharmingen), ICAD (Santa Cruz Biotechnology), α-fodrin (Chemicon International), phospho-ERK, p38, JNK, MEK1/2, ELK, and p90RSK (Cell Signaling Technologies).

    Techniques: Marker, Activity Assay, Positive Control

    Fragmentation of mitochondria without cytochrome c efflux upon productive EMCV infection. Mock-infected and EMCV-infected HeLa cells were incubated for 5 h, immunostained for cytochrome c , and stained for DNA with Hoechst-33342. Alterations in the nuclear

    Journal:

    Article Title: Antiapoptotic Activity of the Cardiovirus Leader Protein, a Viral "Security" Protein ▿

    doi: 10.1128/JVI.00467-09

    Figure Lengend Snippet: Fragmentation of mitochondria without cytochrome c efflux upon productive EMCV infection. Mock-infected and EMCV-infected HeLa cells were incubated for 5 h, immunostained for cytochrome c , and stained for DNA with Hoechst-33342. Alterations in the nuclear

    Article Snippet: The incubations with primary (anti-cytochrome c ; Pharmingen) and secondary (anti-mouse IgG-Alexa 546 conjugate; Invitrogen) antibodies were performed at 37°C in buffer for immunofluorescence (0.1% BSA, 0.05% Tween-20 in PBS).

    Techniques: Infection, Incubation, Staining

    Fragmentation of mitochondria and cytoplasmic exit of cytochrome c in cells infected with the L mutant. (A) HeLa cells expressing mitochondria-targeted EYFP (EYFP-mito) were infected with wt and C19A/C22A MVs for 7 h or were treated with 100 μg/ml

    Journal:

    Article Title: Antiapoptotic Activity of the Cardiovirus Leader Protein, a Viral "Security" Protein ▿

    doi: 10.1128/JVI.00467-09

    Figure Lengend Snippet: Fragmentation of mitochondria and cytoplasmic exit of cytochrome c in cells infected with the L mutant. (A) HeLa cells expressing mitochondria-targeted EYFP (EYFP-mito) were infected with wt and C19A/C22A MVs for 7 h or were treated with 100 μg/ml

    Article Snippet: The incubations with primary (anti-cytochrome c ; Pharmingen) and secondary (anti-mouse IgG-Alexa 546 conjugate; Invitrogen) antibodies were performed at 37°C in buffer for immunofluorescence (0.1% BSA, 0.05% Tween-20 in PBS).

    Techniques: Infection, Mutagenesis, Expressing

    D-gal treatment increases mitochondrial cytochrome c release and cleaved caspase-3 in the auditory cortex of rats. (A) Western blot analysis of Cyt c levels in the mitochondrial fraction. (B) Western blot analysis of Cyt c levels in the cytosolic fraction. (C) Western blot analysis of cleaved caspase-3 levels. The relative levels of Mito Cyt c were normalized to VDAC1; the relative levels of Cyto c and cleaved caspase-3 in each group were normalized to β-actin. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Age-associated decline in Nrf2 signaling and associated mtDNA damage may be involved in the degeneration of the auditory cortex: Implications for central presbycusis

    doi: 10.3892/ijmm.2018.3907

    Figure Lengend Snippet: D-gal treatment increases mitochondrial cytochrome c release and cleaved caspase-3 in the auditory cortex of rats. (A) Western blot analysis of Cyt c levels in the mitochondrial fraction. (B) Western blot analysis of Cyt c levels in the cytosolic fraction. (C) Western blot analysis of cleaved caspase-3 levels. The relative levels of Mito Cyt c were normalized to VDAC1; the relative levels of Cyto c and cleaved caspase-3 in each group were normalized to β-actin. * P

    Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Anti-cleaved caspase-3 (1:200; cat. no. 9661; Cell Signaling Technology, Inc., Danvers, MA, USA) and anti-cytochrome c (1:500; cat. no. ab13575; Abcam).

    Techniques: Western Blot

    TM treatment does not alter copper-dependent enzyme activity. Results are mean enzyme activity ± SE in brain homogenates from the prevention cohort for (a) cytochrome c oxidase (cytocox) and (b) superoxide dismutase (SOD). wt veh = wild type mice

    Journal: Journal of Alzheimer's disease : JAD

    Article Title: A copper-lowering strategy attenuates amyloid pathology in a transgenic mouse model of Alzheimer's disease

    doi: 10.3233/JAD-2010-100408

    Figure Lengend Snippet: TM treatment does not alter copper-dependent enzyme activity. Results are mean enzyme activity ± SE in brain homogenates from the prevention cohort for (a) cytochrome c oxidase (cytocox) and (b) superoxide dismutase (SOD). wt veh = wild type mice

    Article Snippet: Cytochrome c oxidase activity was determined by measuring the oxidation of ferricytochrome c using the Cytochrome c Oxidase Assay Kit (Sigma, St. Louis, Mo) following manufacturer’s instructions.

    Techniques: Activity Assay, Mouse Assay

    Schematic representation of the plausible molecular mechanism proposed for the induction of apoptosis by maslinic acid in HT29 colon-cancer cells . This molecular mechanism is regulated via the induction of JNK and p53, resulting in mitochondrial disruption, the release of cytochrome-c and finally the activation of a cascade of caspases.

    Journal: BMC Cancer

    Article Title: The natural triterpene maslinic acid induces apoptosis in HT29 colon cancer cells by a JNK-p53-dependent mechanism

    doi: 10.1186/1471-2407-11-154

    Figure Lengend Snippet: Schematic representation of the plausible molecular mechanism proposed for the induction of apoptosis by maslinic acid in HT29 colon-cancer cells . This molecular mechanism is regulated via the induction of JNK and p53, resulting in mitochondrial disruption, the release of cytochrome-c and finally the activation of a cascade of caspases.

    Article Snippet: Anti-Bax, anti-Bcl-2, anti-cytochrome-c and secondary antibodies were from Santa Cruz Biotechnology (California, USA).

    Techniques: Activation Assay

    Western blottings of the levels of Bax (panel A), Bcl-2 (panel B) and cytochrome-c (panel C) proteins . HT29 cells were treated with maslinic acid at concentrations of IC 50 and IC 80 for 48 and 72 h. The levels of protein expression are expressed as arbitrary intensity units of each band compared to arbitrary intensity units of actin. The variations in relative percentages of the expression of Bax, Bcl-2 and cytochrome-c for each time and concentration are also shown. The values represent means ± SD of at least three separate experiments.

    Journal: BMC Cancer

    Article Title: The natural triterpene maslinic acid induces apoptosis in HT29 colon cancer cells by a JNK-p53-dependent mechanism

    doi: 10.1186/1471-2407-11-154

    Figure Lengend Snippet: Western blottings of the levels of Bax (panel A), Bcl-2 (panel B) and cytochrome-c (panel C) proteins . HT29 cells were treated with maslinic acid at concentrations of IC 50 and IC 80 for 48 and 72 h. The levels of protein expression are expressed as arbitrary intensity units of each band compared to arbitrary intensity units of actin. The variations in relative percentages of the expression of Bax, Bcl-2 and cytochrome-c for each time and concentration are also shown. The values represent means ± SD of at least three separate experiments.

    Article Snippet: Anti-Bax, anti-Bcl-2, anti-cytochrome-c and secondary antibodies were from Santa Cruz Biotechnology (California, USA).

    Techniques: Western Blot, Expressing, Concentration Assay

    The current model for the potentiation of TRAIL-induced apoptosis in cancer cells by depolarization . Triggering of TRAIL-R1 (DR4)/TRAIL-R2 (DR5) induces the generation and accumulation of mROS, leading to impairment of the ETC function and cardiolipin oxidation. Impairment of the ETC complex I/III function decreases H + efflux, thereby causing ΔΨ m dissipation, i.e., MMD, and additional mROS generation and cardiolipin oxidation, thereby forming a positive loop. Cardiolipin oxidation and ΔΨ m dissipation cooperatively promote the MPT and liberation of sufficient cytochrome c to trigger caspase activation and induce apoptosis. TRAIL-resistant cancer cells appear to gain considerable tolerance for oxidative stress-mediated activation of the intrinsic death pathway. Accumulation of mROS can also promote the formation of unfolded or misfolded proteins, thereby provoking ERS responses such as activation of the transcriptional factor XBP-1. XBP-1 activation leads to upregulation of the surface TRAIL-R2 expression level, thereby enhancing the death signaling. Activation of this alternative death pathway may contribute to commit TRAIL-resistant cancer cells to apoptosis.

    Journal: Frontiers in Oncology

    Article Title: Depolarization Controls TRAIL-Sensitization and Tumor-Selective Killing of Cancer Cells: Crosstalk with ROS

    doi: 10.3389/fonc.2014.00128

    Figure Lengend Snippet: The current model for the potentiation of TRAIL-induced apoptosis in cancer cells by depolarization . Triggering of TRAIL-R1 (DR4)/TRAIL-R2 (DR5) induces the generation and accumulation of mROS, leading to impairment of the ETC function and cardiolipin oxidation. Impairment of the ETC complex I/III function decreases H + efflux, thereby causing ΔΨ m dissipation, i.e., MMD, and additional mROS generation and cardiolipin oxidation, thereby forming a positive loop. Cardiolipin oxidation and ΔΨ m dissipation cooperatively promote the MPT and liberation of sufficient cytochrome c to trigger caspase activation and induce apoptosis. TRAIL-resistant cancer cells appear to gain considerable tolerance for oxidative stress-mediated activation of the intrinsic death pathway. Accumulation of mROS can also promote the formation of unfolded or misfolded proteins, thereby provoking ERS responses such as activation of the transcriptional factor XBP-1. XBP-1 activation leads to upregulation of the surface TRAIL-R2 expression level, thereby enhancing the death signaling. Activation of this alternative death pathway may contribute to commit TRAIL-resistant cancer cells to apoptosis.

    Article Snippet: Permeabilization of the OMM by pro-apoptotic Bcl-2 family proteins promotes the release of a number of apoptogenic factors, such as cytochrome c , endonuclease G, second mitochondrial activator of caspases (SMAC), Omi/HtrA2, and apoptosis-inducing factor (AIF), from the inner mitochondrial membrane (IMM) space into the cytosol, and these apoptogenic proteins promote activation of the caspase cascade, thereby leading to apoptosis.

    Techniques: Activation Assay, Expressing

    IL-2 activated the mitochondrial apoptotic pathway in the presence of sorafenib. a , b Mitochondrial ROS production was detected in HepG2 cells. IL-2 (5 ng/ml) treatment was carried out in the presence of 5 μM sorafenib. c – e The antioxidants in HepG2 cells under IL-2 and sorafenib co-treatment were measured via ELISA. f The mPTP opening rate was analysed to determine the mitochondrial damage. IL-2 (5 ng/ml) treatment was carried out in the presence of 5 μM sorafenib. g , h Cyt-c liberation was observed via immunofluorescence. i – o Mitochondrial apoptotic proteins were analysed by western blotting. The sorafenib-mediated upregulation of apoptotic proteins was further augmented by IL-2 treatment. *P

    Journal: Cancer Cell International

    Article Title: IL-2 augments the sorafenib-induced apoptosis in liver cancer by promoting mitochondrial fission and activating the JNK/TAZ pathway

    doi: 10.1186/s12935-018-0671-3

    Figure Lengend Snippet: IL-2 activated the mitochondrial apoptotic pathway in the presence of sorafenib. a , b Mitochondrial ROS production was detected in HepG2 cells. IL-2 (5 ng/ml) treatment was carried out in the presence of 5 μM sorafenib. c – e The antioxidants in HepG2 cells under IL-2 and sorafenib co-treatment were measured via ELISA. f The mPTP opening rate was analysed to determine the mitochondrial damage. IL-2 (5 ng/ml) treatment was carried out in the presence of 5 μM sorafenib. g , h Cyt-c liberation was observed via immunofluorescence. i – o Mitochondrial apoptotic proteins were analysed by western blotting. The sorafenib-mediated upregulation of apoptotic proteins was further augmented by IL-2 treatment. *P

    Article Snippet: The membranes were then incubated at 4 °C overnight with the primary antibodies [CXCR4 (1:1000, Abcam, #ab1670), CXCR7 (1:1000, Abcam, #ab38089), cyclin D1 (1:1000, Abcam, #ab134175), PCNA (1:1000, Abcam, #ab18197), CDK4 (1:1000, Abcam, #ab137675), cadherin (1:1000, Abcam, #ab133168), vimentin (1:1000, Abcam, #ab8978), TAZ (1:1000, Abcam, #ab224239), complex III subunit core (CIII-core2, 1:1000, Invitrogen, #459220), complex II (CII-30, 1:1000, Abcam, #ab110410), complex IV subunit II (CIV-II, 1:1000, Abcam, #ab110268), Drp1 (1:1000, Abcam, #ab56788), Fis1 (1:1000, Abcam, #ab71498), Opa1 (1:1000, Abcam, #ab42364), Mfn1 (1:1000, Abcam, #ab57602), Mff (1:1000, Cell Signaling Technology, #86668), Bcl2 (1:1000, Cell Signaling Technology, #3498), Bax (1:1000, Cell Signaling Technology, #2772), caspase-9 (1:1000, Cell Signaling Technology, #9504), Bad (1:1000; Abcam; #ab90435), Tom20 (1:1000, Abcam, #ab186735), cyt-c (1:1000; Abcam; #ab90529), GAPDH (1:1000, Cell Signaling Technology, #5174), JNK (1:1000; Cell Signaling Technology, #4672), and p-JNK (1:1000; Cell Signaling Technology, #9251)].

    Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Western Blot

    AS-IV inhibited Aβ1-42-induced cytochrome c release from mitochondria in SK-N-SH cells. A. A representative blots of immunoreactive bands for cytochrome c in cytosol. B. Data were expressed as fold-increase of cytochrome c relative to vehicle. Protein expression levels were normalized to β-actin. C. A representative blots of immunoreactive bands for cytochrome c in mitochondria. D. Data were expressed as fold-increase of cytochrome c relative to vehicle. Protein expression levels were normalized to COX IV. # P

    Journal: PLoS ONE

    Article Title: Protective Effects of Astragaloside IV against Amyloid Beta1-42 Neurotoxicity by Inhibiting the Mitochondrial Permeability Transition Pore Opening

    doi: 10.1371/journal.pone.0098866

    Figure Lengend Snippet: AS-IV inhibited Aβ1-42-induced cytochrome c release from mitochondria in SK-N-SH cells. A. A representative blots of immunoreactive bands for cytochrome c in cytosol. B. Data were expressed as fold-increase of cytochrome c relative to vehicle. Protein expression levels were normalized to β-actin. C. A representative blots of immunoreactive bands for cytochrome c in mitochondria. D. Data were expressed as fold-increase of cytochrome c relative to vehicle. Protein expression levels were normalized to COX IV. # P

    Article Snippet: Cytochrome c oxidase subunit IV (COX IV, Cell signaling) and polyclonal mouse anti β-actin (Sigma) were used as loading controls.

    Techniques: Expressing

    Colocalization of cytochrome c and mitochondria in blastocysts cultured for 7 days in the absence or presence of E-64. (A) Blastocysts were labeled with a cytochrome c -specific antibody (green) and MitoTracker Red (red). (B) Colocalization was analyzed using Image-Pro Plus, Arrow and frame, released cytochrome c .

    Journal: The Journal of Reproduction and Development

    Article Title: Inhibition of cathepsin B activity reduces apoptosis by preventing cytochrome c release from mitochondria in porcine parthenotes

    doi: 10.1262/jrd.2015-019

    Figure Lengend Snippet: Colocalization of cytochrome c and mitochondria in blastocysts cultured for 7 days in the absence or presence of E-64. (A) Blastocysts were labeled with a cytochrome c -specific antibody (green) and MitoTracker Red (red). (B) Colocalization was analyzed using Image-Pro Plus, Arrow and frame, released cytochrome c .

    Article Snippet: Next, the blastocysts were permeabilized as described above and incubated with blocking solution at 37 C for 1 h. The samples were then incubated with an anti-cytochrome c antibody (diluted 1:100; ab110325, Abcam) at 37 C for 2 h. After incubation with primary antibodies, all samples were exposed to a fluorescein isothiocyanate-labeled secondary antibody for 1 h at 37 C. Thereafter, they were washed twice with PVA-PBS, treated with 5 μg/ml Hoechst 33342 for 10 min, washed twice more, and then mounted on glass slides.

    Techniques: Cell Culture, Labeling

    Structures of receptors and proteins (together with relevant properties) described in this work (a) highly functionalized ruthenium tris-bipyridine receptors 1–3 (b) cytochrome c (c) 60% acetylated cytochrome c (d) lysozyme (PDB

    Journal: Organic & biomolecular chemistry

    Article Title: Protein destabilisation by ruthenium(II) tris-bipyridine based protein-surface mimetics, †

    doi: 10.1039/c3ob26251k

    Figure Lengend Snippet: Structures of receptors and proteins (together with relevant properties) described in this work (a) highly functionalized ruthenium tris-bipyridine receptors 1–3 (b) cytochrome c (c) 60% acetylated cytochrome c (d) lysozyme (PDB

    Article Snippet: Likewise, stock solutions of acetylated horse heart or equine cytochrome c (all obtained from Sigma and used without further purification) was prepared to a concentration of ~1 mM in 5 mM sodium phosphate buffer and the pH adjusted to 7.4 via the addition of 1 N sodium hydroxide or 1 N HCl and diluted appropriately.

    Techniques: