caspase 3 levels Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore caspase 3 levels caspase 3 levels
    <t>Caspase-3</t> levels effected by single and combined drugs. Columns, average of six wells. Data are representative of separate three experiments. C, control; IM, imatinib mesylate; LiCl, lithium chloride; IM+LiCl, the combination of IM and LiCl; MPA, medroxyprogesterone acetate; IM+MPA, the combination of IM and MPA.
    Caspase 3 Levels Caspase 3 Levels, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase 3 levels caspase 3 levels/product/Millipore
    Average 99 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    caspase 3 levels caspase 3 levels - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    94
    BioVision caspase 3 levels
    Retinal <t>caspase</t> 3 levels of the groups The box represents 50% of the sample. The remaining 50% of the sample is contained within the areas between the box and the whiskers, with some exceptions (outliers). Single line inside the box represents median. Small circle and asterisk represents the outlier with its number in bevacizumab group and diabetes group, respectively.
    Caspase 3 Levels, supplied by BioVision, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase 3 levels/product/BioVision
    Average 94 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    caspase 3 levels - by Bioz Stars, 2020-08
    94/100 stars
      Buy from Supplier

    90
    USCN Life caspase 3 levels
    Retinal <t>caspase</t> 3 levels of the groups The box represents 50% of the sample. The remaining 50% of the sample is contained within the areas between the box and the whiskers, with some exceptions (outliers). Single line inside the box represents median. Small circle and asterisk represents the outlier with its number in bevacizumab group and diabetes group, respectively.
    Caspase 3 Levels, supplied by USCN Life, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase 3 levels/product/USCN Life
    Average 90 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    caspase 3 levels - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    90
    Becton Dickinson caspase 3 levels
    Endogenous FAIM-L participates in axonal degeneration in DRG neurons. ( A ) Dissociated DRG neurons were plated in Campenot chambers, and distal compartments were filled with medium containing 75 ng/ml human NGF. After infection with shRNAs of scrambled or FAIM-L, local axonal degeneration was induced in one of the axonal compartments, as described in materials and methods. Representative confocal images obtained at the indicated times after NGF deprivation are shown. Images are representative of three independent experiments. Scale bar 10 μm. ( B ) DRG cultures were infected with the different lentiviral vectors as indicated, and NGF was withdrawn for 24 h. <t>Caspase-3</t> (pro-form p32), cleaved caspase-3 (active form p17) and NF-66 levels were assessed at the indicated times by Western blot. ERK was used as loading control. ( C ) Histogram representing active caspase-3 levels relative to total caspase-3 and NF-66 cleaved fragment relative to total NF-66 in each condition 24 h after NGF withdrawal. Data are represented as the mean ± SEM of three independent experiments. One-way ANOVA with Tukey’s multiple comparison post-hoc test was used to calculate significant levels between the indicated groups. *p
    Caspase 3 Levels, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase 3 levels/product/Becton Dickinson
    Average 90 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    caspase 3 levels - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    99
    Millipore caspase 3
    Western blotting analysis of the expression levels of <t>caspase-3</t> and Bcl-2. The expression level of caspase-3 was reduced by allantoin (A), while Bcl2 expression was increased (B) ( n = 6 for each group). Data are presented as the mean ± SE. ∗ P
    Caspase 3, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2538 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase 3/product/Millipore
    Average 99 stars, based on 2538 article reviews
    Price from $9.99 to $1999.99
    caspase 3 - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc casp3 caspase 3
    Increased apoptosis in KO myotubes. ( A ) Western blot of total lysates (top) and nuclear and mitochondrial fractions of WT and KO myotubes grown in differentiation medium for 6 to 7 d. No changes in the levels of activated (cleaved) <t>CASP3</t> products are
    Casp3 Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/casp3 caspase 3/product/Cell Signaling Technology Inc
    Average 99 stars, based on 80 article reviews
    Price from $9.99 to $1999.99
    casp3 caspase 3 - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc caspase 3
    Effect of lidocaine on hypoxia-induced apoptosis of H9c2 cells. Cell apoptosis was measured using a FITC-Annexin V/PI detection kit. (A) Representative images of flow cytometry data and (B) apoptosis rates. (C) Representative images of western blotting analysis of Bax, Bcl-2 and <t>caspase-3.</t> **P
    Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 27981 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase 3/product/Cell Signaling Technology Inc
    Average 99 stars, based on 27981 article reviews
    Price from $9.99 to $1999.99
    caspase 3 - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    95
    Cell Signaling Technology Inc anti cleaved caspase 3
    DUSP6 SUMOylation at K234 is protective to brain neurons in ischemic stroke. ( A ) Brain ischemia and brain damage in the tMCAO model. Left: Regional cerebral blood flow in the core of the middle cerebral artery territory of C57BL/6 mice before, during, and after ischemia as measured by LDF. Flow values were averaged at 5-min intervals and expressed as means ± SEM of nine experiments. Right: Representative photographs of TTC-stained brain sections (1 mm) showing brain infarction at 24 hours after the onset of ischemia. ( B ) Time-dependent decreases in DUSP6 SUMOylation and DUSP6 protein levels during reperfusion in mice subjected to tMCAO for 40 min. Cerebral cortices from C57BL/6 mice (wild type) that underwent 40 min tMCAO, followed by different periods of reperfusion as indicated, were lysed under denaturing conditions and subjected to IP with anti-DUSP6 (or the IgG control), which was followed by IB using anti-SUMO1 and anti-DUSP6. β-Actin was used as the loading control. Note also the time-dependent decrease in overall protein SUMOylation. ( C to G ) Transcriptional up-regulation of SENP1 during reperfusion of tMCAO injured mice. The original lysates as in (B) were analyzed by real-time quantitative RT-PCR for mRNA levels of DUSP6 (C), SAE1/SAE2 (D), UBC9 (E), and SENP1 (F), as well as by IB for protein levels of SAE1, SAE2, UBC9, and SENP1, with β-actin as the loading control (G). Only SENP1 showed time-dependent increases at both the mRNA (F) and protein levels (G). ( H ) Schematic representation of adeno-associated virus (AAV) infection into mouse primary somatosensory cortex, barrel field, and secondary somatosensory cortex (left) and representative brain images showing the expression of mNeonGreen in cerebral cortex (right). The boxed area is enhanced by high magnification to show mNeonGreen-positive neurons. Scale bar, 100 μm. ( I to K ) Overexpression of wild-type DUSP6 reduced, but the SUMOylation-deficient DUSP6 K234R mutant exacerbated, cortical neuron apoptosis in response to brain I/R. AAV-DUSP6, AAV-DUSP6 K234R , or a control AAV driven by the human synapsin promoter (hSyn) was injected into the primary somatosensory cortex, barrel field, and secondary somatosensory cortex of adult wild-type C57BL/6 mice as shown in (H). The animals were subjected to 40-min tMCAO, followed by 24-hour reperfusion at 4 weeks after stereotactic intracerebral injection. (I) The cerebral cortical tissues were lysed and subjected to IB using anti-cleaved <t>caspase-3,</t> anti–p–Drp1-S616, anti–p–Drp1-S637, anti-Drp1, anti-Flag, and anti-DUSP6. β-Actin was used as the loading control. Caspase-3 cleavage was reduced by AAV-DUSP6 but increased by AAV-DUSP6 K234R . Note also the corresponding changes in p–Drp1-S616 levels; however, p–Drp1-S637 levels were affected by neither AAV-DUSP6 nor AAV-DUSP6 K234R , despite the decrease caused by I/R. ( J ) The tissues were sectioned and stained by TUNEL (red) and DAPI (blue). The occurrence of TUNEL + cells in areas with DUSP6 expression (green) was significantly less than that with DUSP6 K234R expression. Representative confocal images in virus-infected cortical areas of brain sections are shown at the left. Scale bar, 100 μm. Quantification of TUNEL + cells are shown at the right. Data represent means ± SEM of n = 3 independent experiments, with six to eight fields of each group used for statistics in each experiment. * P
    Anti Cleaved Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 11190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti cleaved caspase 3/product/Cell Signaling Technology Inc
    Average 95 stars, based on 11190 article reviews
    Price from $9.99 to $1999.99
    anti cleaved caspase 3 - by Bioz Stars, 2020-08
    95/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc polyclonal anti cleaved caspase 3
    DUSP6 SUMOylation at K234 is protective to brain neurons in ischemic stroke. ( A ) Brain ischemia and brain damage in the tMCAO model. Left: Regional cerebral blood flow in the core of the middle cerebral artery territory of C57BL/6 mice before, during, and after ischemia as measured by LDF. Flow values were averaged at 5-min intervals and expressed as means ± SEM of nine experiments. Right: Representative photographs of TTC-stained brain sections (1 mm) showing brain infarction at 24 hours after the onset of ischemia. ( B ) Time-dependent decreases in DUSP6 SUMOylation and DUSP6 protein levels during reperfusion in mice subjected to tMCAO for 40 min. Cerebral cortices from C57BL/6 mice (wild type) that underwent 40 min tMCAO, followed by different periods of reperfusion as indicated, were lysed under denaturing conditions and subjected to IP with anti-DUSP6 (or the IgG control), which was followed by IB using anti-SUMO1 and anti-DUSP6. β-Actin was used as the loading control. Note also the time-dependent decrease in overall protein SUMOylation. ( C to G ) Transcriptional up-regulation of SENP1 during reperfusion of tMCAO injured mice. The original lysates as in (B) were analyzed by real-time quantitative RT-PCR for mRNA levels of DUSP6 (C), SAE1/SAE2 (D), UBC9 (E), and SENP1 (F), as well as by IB for protein levels of SAE1, SAE2, UBC9, and SENP1, with β-actin as the loading control (G). Only SENP1 showed time-dependent increases at both the mRNA (F) and protein levels (G). ( H ) Schematic representation of adeno-associated virus (AAV) infection into mouse primary somatosensory cortex, barrel field, and secondary somatosensory cortex (left) and representative brain images showing the expression of mNeonGreen in cerebral cortex (right). The boxed area is enhanced by high magnification to show mNeonGreen-positive neurons. Scale bar, 100 μm. ( I to K ) Overexpression of wild-type DUSP6 reduced, but the SUMOylation-deficient DUSP6 K234R mutant exacerbated, cortical neuron apoptosis in response to brain I/R. AAV-DUSP6, AAV-DUSP6 K234R , or a control AAV driven by the human synapsin promoter (hSyn) was injected into the primary somatosensory cortex, barrel field, and secondary somatosensory cortex of adult wild-type C57BL/6 mice as shown in (H). The animals were subjected to 40-min tMCAO, followed by 24-hour reperfusion at 4 weeks after stereotactic intracerebral injection. (I) The cerebral cortical tissues were lysed and subjected to IB using anti-cleaved <t>caspase-3,</t> anti–p–Drp1-S616, anti–p–Drp1-S637, anti-Drp1, anti-Flag, and anti-DUSP6. β-Actin was used as the loading control. Caspase-3 cleavage was reduced by AAV-DUSP6 but increased by AAV-DUSP6 K234R . Note also the corresponding changes in p–Drp1-S616 levels; however, p–Drp1-S637 levels were affected by neither AAV-DUSP6 nor AAV-DUSP6 K234R , despite the decrease caused by I/R. ( J ) The tissues were sectioned and stained by TUNEL (red) and DAPI (blue). The occurrence of TUNEL + cells in areas with DUSP6 expression (green) was significantly less than that with DUSP6 K234R expression. Representative confocal images in virus-infected cortical areas of brain sections are shown at the left. Scale bar, 100 μm. Quantification of TUNEL + cells are shown at the right. Data represent means ± SEM of n = 3 independent experiments, with six to eight fields of each group used for statistics in each experiment. * P
    Polyclonal Anti Cleaved Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal anti cleaved caspase 3/product/Cell Signaling Technology Inc
    Average 99 stars, based on 87 article reviews
    Price from $9.99 to $1999.99
    polyclonal anti cleaved caspase 3 - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Promega caspase 3 7 levels
    DUSP6 SUMOylation at K234 is protective to brain neurons in ischemic stroke. ( A ) Brain ischemia and brain damage in the tMCAO model. Left: Regional cerebral blood flow in the core of the middle cerebral artery territory of C57BL/6 mice before, during, and after ischemia as measured by LDF. Flow values were averaged at 5-min intervals and expressed as means ± SEM of nine experiments. Right: Representative photographs of TTC-stained brain sections (1 mm) showing brain infarction at 24 hours after the onset of ischemia. ( B ) Time-dependent decreases in DUSP6 SUMOylation and DUSP6 protein levels during reperfusion in mice subjected to tMCAO for 40 min. Cerebral cortices from C57BL/6 mice (wild type) that underwent 40 min tMCAO, followed by different periods of reperfusion as indicated, were lysed under denaturing conditions and subjected to IP with anti-DUSP6 (or the IgG control), which was followed by IB using anti-SUMO1 and anti-DUSP6. β-Actin was used as the loading control. Note also the time-dependent decrease in overall protein SUMOylation. ( C to G ) Transcriptional up-regulation of SENP1 during reperfusion of tMCAO injured mice. The original lysates as in (B) were analyzed by real-time quantitative RT-PCR for mRNA levels of DUSP6 (C), SAE1/SAE2 (D), UBC9 (E), and SENP1 (F), as well as by IB for protein levels of SAE1, SAE2, UBC9, and SENP1, with β-actin as the loading control (G). Only SENP1 showed time-dependent increases at both the mRNA (F) and protein levels (G). ( H ) Schematic representation of adeno-associated virus (AAV) infection into mouse primary somatosensory cortex, barrel field, and secondary somatosensory cortex (left) and representative brain images showing the expression of mNeonGreen in cerebral cortex (right). The boxed area is enhanced by high magnification to show mNeonGreen-positive neurons. Scale bar, 100 μm. ( I to K ) Overexpression of wild-type DUSP6 reduced, but the SUMOylation-deficient DUSP6 K234R mutant exacerbated, cortical neuron apoptosis in response to brain I/R. AAV-DUSP6, AAV-DUSP6 K234R , or a control AAV driven by the human synapsin promoter (hSyn) was injected into the primary somatosensory cortex, barrel field, and secondary somatosensory cortex of adult wild-type C57BL/6 mice as shown in (H). The animals were subjected to 40-min tMCAO, followed by 24-hour reperfusion at 4 weeks after stereotactic intracerebral injection. (I) The cerebral cortical tissues were lysed and subjected to IB using anti-cleaved <t>caspase-3,</t> anti–p–Drp1-S616, anti–p–Drp1-S637, anti-Drp1, anti-Flag, and anti-DUSP6. β-Actin was used as the loading control. Caspase-3 cleavage was reduced by AAV-DUSP6 but increased by AAV-DUSP6 K234R . Note also the corresponding changes in p–Drp1-S616 levels; however, p–Drp1-S637 levels were affected by neither AAV-DUSP6 nor AAV-DUSP6 K234R , despite the decrease caused by I/R. ( J ) The tissues were sectioned and stained by TUNEL (red) and DAPI (blue). The occurrence of TUNEL + cells in areas with DUSP6 expression (green) was significantly less than that with DUSP6 K234R expression. Representative confocal images in virus-infected cortical areas of brain sections are shown at the left. Scale bar, 100 μm. Quantification of TUNEL + cells are shown at the right. Data represent means ± SEM of n = 3 independent experiments, with six to eight fields of each group used for statistics in each experiment. * P
    Caspase 3 7 Levels, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/caspase 3 7 levels/product/Promega
    Average 99 stars, based on 42 article reviews
    Price from $9.99 to $1999.99
    caspase 3 7 levels - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    Image Search Results


    Caspase-3 levels effected by single and combined drugs. Columns, average of six wells. Data are representative of separate three experiments. C, control; IM, imatinib mesylate; LiCl, lithium chloride; IM+LiCl, the combination of IM and LiCl; MPA, medroxyprogesterone acetate; IM+MPA, the combination of IM and MPA.

    Journal: Journal of Gynecologic Oncology

    Article Title: Combination of imatinib mesylate with lithium chloride and medroxyprogesterone acetate is highly active in Ishikawa endometrial carcinoma in vitro

    doi: 10.3802/jgo.2011.22.4.225

    Figure Lengend Snippet: Caspase-3 levels effected by single and combined drugs. Columns, average of six wells. Data are representative of separate three experiments. C, control; IM, imatinib mesylate; LiCl, lithium chloride; IM+LiCl, the combination of IM and LiCl; MPA, medroxyprogesterone acetate; IM+MPA, the combination of IM and MPA.

    Article Snippet: Caspase-3 levels Caspase-3 levels in triplicate were analyzed using fluorimetric kits (Sigma Aldrich, St. Louis, MO, USA).

    Techniques:

    Effects of different concentrations (1, 5, 10, 50 IU/ml) of erythropoietin (EPO) treatment, glibenclamide (Gli 10,100 μM) and diazoxide (Dia 10,100 μM) on apoptosis in normoxic conditions at 2, 24 and 48 h by the caspase-3 activity levels. Data are presented as mean ± SE (n=40 observations).

    Journal: The Indian Journal of Medical Research

    Article Title: Role of ATP-dependent K channels in the effects of erythropoietin in renal ischaemia injury

    doi: 10.4103/0971-5916.160713

    Figure Lengend Snippet: Effects of different concentrations (1, 5, 10, 50 IU/ml) of erythropoietin (EPO) treatment, glibenclamide (Gli 10,100 μM) and diazoxide (Dia 10,100 μM) on apoptosis in normoxic conditions at 2, 24 and 48 h by the caspase-3 activity levels. Data are presented as mean ± SE (n=40 observations).

    Article Snippet: Caspase-3 levels were evaluated in EPO, Gli and/or Dia treated cells at the 48 h. Cell proliferation was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT, Sigma, USA) at the 2, 24 and 48 h as mentioned in previous study .

    Techniques: Activity Assay

    Effects of different concentrations (1,5,10,50 IU/ml) of erythropoietin (EPO) treatment, glibenclamide (Gli 10,100 μM) and diazoxide (Dia 10,100 μM) on apoptosis in hypoxic conditions at 2, 24 and 48 h by the caspase-3 activity levels. Data are presented as mean ± SE (n=40 observations).

    Journal: The Indian Journal of Medical Research

    Article Title: Role of ATP-dependent K channels in the effects of erythropoietin in renal ischaemia injury

    doi: 10.4103/0971-5916.160713

    Figure Lengend Snippet: Effects of different concentrations (1,5,10,50 IU/ml) of erythropoietin (EPO) treatment, glibenclamide (Gli 10,100 μM) and diazoxide (Dia 10,100 μM) on apoptosis in hypoxic conditions at 2, 24 and 48 h by the caspase-3 activity levels. Data are presented as mean ± SE (n=40 observations).

    Article Snippet: Caspase-3 levels were evaluated in EPO, Gli and/or Dia treated cells at the 48 h. Cell proliferation was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT, Sigma, USA) at the 2, 24 and 48 h as mentioned in previous study .

    Techniques: Activity Assay

    Retinal caspase 3 levels of the groups The box represents 50% of the sample. The remaining 50% of the sample is contained within the areas between the box and the whiskers, with some exceptions (outliers). Single line inside the box represents median. Small circle and asterisk represents the outlier with its number in bevacizumab group and diabetes group, respectively.

    Journal: International Journal of Ophthalmology

    Article Title: Comparison of the effect of intravitreal bevacizumab and intravitreal fasudil on retinal VEGF, TNF?, and caspase 3 levels in an experimental diabetes model

    doi: 10.3980/j.issn.2222-3959.2014.01.10

    Figure Lengend Snippet: Retinal caspase 3 levels of the groups The box represents 50% of the sample. The remaining 50% of the sample is contained within the areas between the box and the whiskers, with some exceptions (outliers). Single line inside the box represents median. Small circle and asterisk represents the outlier with its number in bevacizumab group and diabetes group, respectively.

    Article Snippet: The VEGF and TNFα levels were estimated with kits from Cusabio Biotech Co., Ltd. (Wuhan, China), and the caspase 3 levels were estimated with a kit from BioVision Inc. (California, USA) according to the manufacturers' instructions.

    Techniques:

    Endogenous FAIM-L participates in axonal degeneration in DRG neurons. ( A ) Dissociated DRG neurons were plated in Campenot chambers, and distal compartments were filled with medium containing 75 ng/ml human NGF. After infection with shRNAs of scrambled or FAIM-L, local axonal degeneration was induced in one of the axonal compartments, as described in materials and methods. Representative confocal images obtained at the indicated times after NGF deprivation are shown. Images are representative of three independent experiments. Scale bar 10 μm. ( B ) DRG cultures were infected with the different lentiviral vectors as indicated, and NGF was withdrawn for 24 h. Caspase-3 (pro-form p32), cleaved caspase-3 (active form p17) and NF-66 levels were assessed at the indicated times by Western blot. ERK was used as loading control. ( C ) Histogram representing active caspase-3 levels relative to total caspase-3 and NF-66 cleaved fragment relative to total NF-66 in each condition 24 h after NGF withdrawal. Data are represented as the mean ± SEM of three independent experiments. One-way ANOVA with Tukey’s multiple comparison post-hoc test was used to calculate significant levels between the indicated groups. *p

    Journal: Scientific Reports

    Article Title: FAIM-L regulation of XIAP degradation modulates Synaptic Long-Term Depression and Axon Degeneration

    doi: 10.1038/srep35775

    Figure Lengend Snippet: Endogenous FAIM-L participates in axonal degeneration in DRG neurons. ( A ) Dissociated DRG neurons were plated in Campenot chambers, and distal compartments were filled with medium containing 75 ng/ml human NGF. After infection with shRNAs of scrambled or FAIM-L, local axonal degeneration was induced in one of the axonal compartments, as described in materials and methods. Representative confocal images obtained at the indicated times after NGF deprivation are shown. Images are representative of three independent experiments. Scale bar 10 μm. ( B ) DRG cultures were infected with the different lentiviral vectors as indicated, and NGF was withdrawn for 24 h. Caspase-3 (pro-form p32), cleaved caspase-3 (active form p17) and NF-66 levels were assessed at the indicated times by Western blot. ERK was used as loading control. ( C ) Histogram representing active caspase-3 levels relative to total caspase-3 and NF-66 cleaved fragment relative to total NF-66 in each condition 24 h after NGF withdrawal. Data are represented as the mean ± SEM of three independent experiments. One-way ANOVA with Tukey’s multiple comparison post-hoc test was used to calculate significant levels between the indicated groups. *p

    Article Snippet: In DRG neurons, XIAP, FAIM-L, pan-ERK, NF66 and caspase-3 levels were assessed by Western blot using anti-XIAP (BD Biosciences; 1:20,000), anti-FAIM-L (in house; 1:2,000), anti-pan-ERK (BD Biosciences; 1:20,000), anti-NF66 (Covance; 1:10,000), anti-caspase-3 (Cell Signaling; 1:1,000) and Histone H3 (Thermo Scientific; 1:40,000) antibodies.

    Techniques: Infection, Western Blot

    FAIM-L is downregulated in NGF-deprived DRGs and its overexpression impairs axonal DRG degeneration induced by NGF withdrawal. DRG explants were plated in presence of lentiviral vectors carrying EMPTY-EGFP or FAIM-L-EGFP constructs. At DIV 2, DRG explants were subjected to NGF withdrawal. ( A ) at indicated times axonal integrity was assessed by immunocytochemistry against βIII-Tubulin. Representative pictures for EMPT-EGFP- and FAIM-L-EGFP-infected DRGs are shown. Scale bar 200 μm. ( B ) FAIM-L expression, caspase-3 activation, and NF-66 degradation were assessed by Western blot. Histograms show FAIM-L, caspase-3 p17 fragment, and NF-66 cleaved fragment relative levels quantification to Histone H3, total caspase-3 and total NF-66, respectively, from three independent experiments in the indicated conditions. Data are represented as the mean ± standard error of the mean (SEM). Two-way ANOVA test followed by Bonferroni post-hoc test was used to calculate significant levels between the indicated groups. *p

    Journal: Scientific Reports

    Article Title: FAIM-L regulation of XIAP degradation modulates Synaptic Long-Term Depression and Axon Degeneration

    doi: 10.1038/srep35775

    Figure Lengend Snippet: FAIM-L is downregulated in NGF-deprived DRGs and its overexpression impairs axonal DRG degeneration induced by NGF withdrawal. DRG explants were plated in presence of lentiviral vectors carrying EMPTY-EGFP or FAIM-L-EGFP constructs. At DIV 2, DRG explants were subjected to NGF withdrawal. ( A ) at indicated times axonal integrity was assessed by immunocytochemistry against βIII-Tubulin. Representative pictures for EMPT-EGFP- and FAIM-L-EGFP-infected DRGs are shown. Scale bar 200 μm. ( B ) FAIM-L expression, caspase-3 activation, and NF-66 degradation were assessed by Western blot. Histograms show FAIM-L, caspase-3 p17 fragment, and NF-66 cleaved fragment relative levels quantification to Histone H3, total caspase-3 and total NF-66, respectively, from three independent experiments in the indicated conditions. Data are represented as the mean ± standard error of the mean (SEM). Two-way ANOVA test followed by Bonferroni post-hoc test was used to calculate significant levels between the indicated groups. *p

    Article Snippet: In DRG neurons, XIAP, FAIM-L, pan-ERK, NF66 and caspase-3 levels were assessed by Western blot using anti-XIAP (BD Biosciences; 1:20,000), anti-FAIM-L (in house; 1:2,000), anti-pan-ERK (BD Biosciences; 1:20,000), anti-NF66 (Covance; 1:10,000), anti-caspase-3 (Cell Signaling; 1:1,000) and Histone H3 (Thermo Scientific; 1:40,000) antibodies.

    Techniques: Over Expression, Construct, Immunocytochemistry, Infection, Expressing, Activation Assay, Western Blot

    Apoptosis in A549 cells expressing L101P and R280 mutations . A549 cells were transfected with pEYFP-N1/ABCA3 plasmids and apoptotic markers were analysed by FACS in the transfected (YFP + ) population of cells and A549 cells by assaying (A) annexin V + /propidium iodide (PI) - staining, (B) intracellular (i.c.) glutathione (GSH) level through coupling of monochlorobimane with GSH, to determine early apoptosis, and (C) intracellular active caspase 3 level (MFI), to detect late apoptosis. Elevated early and late apoptotic markers were detectable in cells expressing L101P mutation, and one early marker (Annexin V) in cells expressing R280C mutation. Results were calculated from six independent experiments; * p

    Journal: Respiratory Research

    Article Title: Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells

    doi: 10.1186/1465-9921-12-4

    Figure Lengend Snippet: Apoptosis in A549 cells expressing L101P and R280 mutations . A549 cells were transfected with pEYFP-N1/ABCA3 plasmids and apoptotic markers were analysed by FACS in the transfected (YFP + ) population of cells and A549 cells by assaying (A) annexin V + /propidium iodide (PI) - staining, (B) intracellular (i.c.) glutathione (GSH) level through coupling of monochlorobimane with GSH, to determine early apoptosis, and (C) intracellular active caspase 3 level (MFI), to detect late apoptosis. Elevated early and late apoptotic markers were detectable in cells expressing L101P mutation, and one early marker (Annexin V) in cells expressing R280C mutation. Results were calculated from six independent experiments; * p

    Article Snippet: Early apoptosis was assayed by 1) measuring intracellular glutathione (GSH) levels with the cell-permeable monochlorobimane (Sigma) method [ ] and 2) by annexin V+ /propidium iodide (PI)- staining (Cy5-conjugated anti-annexin V and PI; BD Bioscience, Heidelberg, Germany), and late apoptosis was determined via intracellular active caspase 3 levels (PE-conjugated anti-active-caspase 3; BD Bioscience) in cells permeabilized with the IntraPrep kit (Beckman Coulter, Krefeld, Germany) according to the manufacturer's protocol.

    Techniques: Expressing, Transfection, FACS, Staining, Mutagenesis, Marker

    Western blotting analysis of the expression levels of caspase-3 and Bcl-2. The expression level of caspase-3 was reduced by allantoin (A), while Bcl2 expression was increased (B) ( n = 6 for each group). Data are presented as the mean ± SE. ∗ P

    Journal: PeerJ

    Article Title: Allantoin ameliorates chemically-induced pancreatic β-cell damage through activation of the imidazoline I3 receptors

    doi: 10.7717/peerj.1105

    Figure Lengend Snippet: Western blotting analysis of the expression levels of caspase-3 and Bcl-2. The expression level of caspase-3 was reduced by allantoin (A), while Bcl2 expression was increased (B) ( n = 6 for each group). Data are presented as the mean ± SE. ∗ P

    Article Snippet: In the STZ-treated group, the expression level of caspase-3 was significantly increased, while the Bcl-2 level was significantly decreased.

    Techniques: Western Blot, Expressing

    Increased apoptosis in KO myotubes. ( A ) Western blot of total lysates (top) and nuclear and mitochondrial fractions of WT and KO myotubes grown in differentiation medium for 6 to 7 d. No changes in the levels of activated (cleaved) CASP3 products are

    Journal: Autophagy

    Article Title: Defects in calcium homeostasis and mitochondria can be reversed in Pompe disease

    doi: 10.1080/15548627.2015.1009779

    Figure Lengend Snippet: Increased apoptosis in KO myotubes. ( A ) Western blot of total lysates (top) and nuclear and mitochondrial fractions of WT and KO myotubes grown in differentiation medium for 6 to 7 d. No changes in the levels of activated (cleaved) CASP3 products are

    Article Snippet: The following antibodies were used for western blots: VCL/Vinculin (Sigma, V9131), LC3B (Sigma, L8918), CACNB1 (S7–18; Abcam, ab85020), MFN2/mitofusin 2 (Abcam, ab50830), COX4I1/COXIV (Abcam, ab14744), FIS1/TTC11/ Fission 1 (Abcam, ab71498), PARK2/Parkin (Abcam, ab15954), TUBA1A/α-tubulin (Abcam, ab18251), PINK1 (Abcam, ab23707), ubiquitin (linkage-specific K63; Millipore, 05–1308), SQSTM1 (Santa Cruz Biotechnology, sc-25575), CASP3/caspase 3 (Cell Signaling Technology, 9662), AIFM1/AIF (Cell Signaling Technology, 5318), HIST1H3A/ Histone H3 (D1H2; Cell Signaling Technology, 4499), OPA1 (BD Biosciences, 612606), DNM1L/DRP1 (DNM1L; BD Biosciences, 611738), Alexa Fluor-conjugated secondary anti-mouse (Life Technologies, A-21057) or anti-rabbit (Life Technologies, A-21076).

    Techniques: Western Blot

    IL-26 suppressed the apoptosis of HSCs by inhibition of caspase 3 (CASP3) and Bcl-2 associated X protein (BAX). A. Apoptosis of HSCs by annexin V-FITC/PI double staining after treatment with IL-26 at 0, 50, 100, and 200 pg/ml for 48 h. B. The mRNA expression levels of CASP3 and BAX were detected by quantitative real-time PCR (qRT-PCR) in the different concentrations of IL-26 groups. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the reference control. C and D. Western blotting analysis of the protein levels of CASP3, cleaved CASP3, and BAX in HSCs after IL-26 treatment. * P

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Interleukin-26 promotes the proliferation and activation of hepatic stellate cells to exacerbate liver fibrosis by the TGF-β1/Smad2 signaling pathway

    doi:

    Figure Lengend Snippet: IL-26 suppressed the apoptosis of HSCs by inhibition of caspase 3 (CASP3) and Bcl-2 associated X protein (BAX). A. Apoptosis of HSCs by annexin V-FITC/PI double staining after treatment with IL-26 at 0, 50, 100, and 200 pg/ml for 48 h. B. The mRNA expression levels of CASP3 and BAX were detected by quantitative real-time PCR (qRT-PCR) in the different concentrations of IL-26 groups. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the reference control. C and D. Western blotting analysis of the protein levels of CASP3, cleaved CASP3, and BAX in HSCs after IL-26 treatment. * P

    Article Snippet: Membranes were blocked with 5% non-fat milk at 37°C for 2 h and primary mouse monoclonal anti-TGF-β1 (#ab27969, 1:1000; Abcam, Cambridge, MA, USA) and anti-Bcl-2 associated X protein (BAX) (#ab77566, 1:500; Abcam) antibodies, and primary rabbit monoclonal anti-SMAD family member 2 (Smad2) (#ab40855, 1:500; Abcam), anti-caspase 3 (CASP3) (#9662, 1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-cleaved CASP3 (#9661, 1:1500; Cell Signaling Technology) antibodies were prepared in blocking solution and incubated overnight at 4°C.

    Techniques: Inhibition, Double Staining, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

    miR-519a sensitized GBM cells to TMZ treatment partly regulated by autophagy. U87-MG/TMZ cells transfected with miR-519a were treated with or without 400 μM TMZ after incubation with 3-MA (an inhibitor of autophagy) for 2 h. U87-MG cells transfected with anti- miR-519a were treated with or without 200 nM rapamycin for 2 h. The cells were then analyzed for the following: a assessment of proliferation by MTT assay; b , e assessment of colony formation; c , d assessment of apoptosis by FACS analysis of PI-stained cells; and f assessment of caspase-3 activity. * p

    Journal: Journal of Hematology & Oncology

    Article Title: miR-519a enhances chemosensitivity and promotes autophagy in glioblastoma by targeting STAT3/Bcl2 signaling pathway

    doi: 10.1186/s13045-018-0618-0

    Figure Lengend Snippet: miR-519a sensitized GBM cells to TMZ treatment partly regulated by autophagy. U87-MG/TMZ cells transfected with miR-519a were treated with or without 400 μM TMZ after incubation with 3-MA (an inhibitor of autophagy) for 2 h. U87-MG cells transfected with anti- miR-519a were treated with or without 200 nM rapamycin for 2 h. The cells were then analyzed for the following: a assessment of proliferation by MTT assay; b , e assessment of colony formation; c , d assessment of apoptosis by FACS analysis of PI-stained cells; and f assessment of caspase-3 activity. * p

    Article Snippet: The antibodies including anti-LC3B (cat. no. 4445), anti-BECN1/Beclin (cat. no. 3495), anti-STAT3 (cat. no. 12640), anti-phospho-STAT3 (Tyr705; cat. no. 9145), and anti-CASP3/caspase-3 (cat. no. 9915) were purchased from Cell Signaling Technology, while anti-Bax (cat. no. sc-7480) and anti-Bcl-2 (cat. no. sc-509) were obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA.

    Techniques: Transfection, Incubation, MTT Assay, FACS, Staining, Activity Assay

    A : ATPAF1 (target of miR-26a, miR-28–5p, let-7i-5p, and let-7e-5p). B : BACE1 (target of miR-103–3p, miR-374–5p, miR-7a-5p and miR-19a-3p-3p). C : CASP3 (target of miR-103-rp, let-7e-5p and let-7i-5p). D : citrate synthase (CS) (target of miR-19a-3p-3p). E : GPD2 (target of miR-30a-5p). F : LRRK2 (target of miR-19a-3p-3p and miR-181a-5p). G : MAP2K4 (target of miR-24–3p and miR-374–5p). H : VDAC1 (target of miR-7a-5p) total protein content in the TA of HCR and LCR rats ( n = 9). Values are arbitrary units expressed relative to stain-free total protein loading. *Significantly different ( P

    Journal: American Journal of Physiology - Endocrinology and Metabolism

    Article Title: Expression of microRNAs and target proteins in skeletal muscle of rats selectively bred for high and low running capacity

    doi: 10.1152/ajpendo.00043.2017

    Figure Lengend Snippet: A : ATPAF1 (target of miR-26a, miR-28–5p, let-7i-5p, and let-7e-5p). B : BACE1 (target of miR-103–3p, miR-374–5p, miR-7a-5p and miR-19a-3p-3p). C : CASP3 (target of miR-103-rp, let-7e-5p and let-7i-5p). D : citrate synthase (CS) (target of miR-19a-3p-3p). E : GPD2 (target of miR-30a-5p). F : LRRK2 (target of miR-19a-3p-3p and miR-181a-5p). G : MAP2K4 (target of miR-24–3p and miR-374–5p). H : VDAC1 (target of miR-7a-5p) total protein content in the TA of HCR and LCR rats ( n = 9). Values are arbitrary units expressed relative to stain-free total protein loading. *Significantly different ( P

    Article Snippet: Primary antibodies used were polyclonal caspase-3 (CASP3) (no. 9662), leucine-rich repeat kinase 2 (LRRK2) (no. 5559) (Cell Signaling, Beverly, MA), polyclonal ATP synthase mitochondrial F1 complex assembly factor 1 (ATPAF1) (no. ab101518), beta-site APP cleaving enzyme 1 (BACE1; ab2077), citrate synthase (CS; ab96600), monoclonal glycerol-3-phosphate dehydrogenase 2 (GPD2; ab188585), MAP2K4 (ab33912), and VDAC1 (ab14734; Abcam, Cambridge, UK).

    Techniques: Staining

    2-APB attenuates the sevoflurane-induced caspase-3 activation and Aβ accumulation in the brain tissues of neonatal naïve mice A . Anesthesia with 3% sevoflurane for six hours (lanes 2 to 3) induces caspase-3 cleavage (activation) as compared to control condition (lane 1). 2-APB (5 mg/kg: lanes 6 to 8; and 10 mg/kg: lanes 4 to 5) attenuates the sevoflurane-induced caspase-3 activation. B . Quantification of the Western blot shows that the sevoflurane anesthesia (black bar) induces caspase-3 activation compared with control condition (white bar). 2-APB (5 mg/kg: gray bar; and 10 mg/kg: net bar) attenuates the sevoflurane-induced caspase-3 activation (black bar). C . The sevoflurane anesthesia (lanes 3 to 4) increases Aβ levels as compared to control condition (lanes 1 to 2). The 2-APB treatment (lanes 5 to 6) attenuates the sevoflurane-induced increases of Aβ levels (lanes 3 to 4). D . Quantification of the Western blot shows that the sevoflurane anesthesia (black bar) increases Aβ levels compared with control condition (white bar). The 2-APB treatment (10 mg/kg, net bar) attenuates the sevoflurane-induced increase of Aβ (black bar). We have averaged results from four independent experiments. Aβ, β-amyloid protein; FL, full length. * or # P

    Journal: Anesthesiology

    Article Title: Anesthetic Sevoflurane Causes Neurotoxicity Differently in Neonatal Na?ve and Alzheimer's Disease Transgenic Mice

    doi: 10.1097/ALN.0b013e3181d94de1

    Figure Lengend Snippet: 2-APB attenuates the sevoflurane-induced caspase-3 activation and Aβ accumulation in the brain tissues of neonatal naïve mice A . Anesthesia with 3% sevoflurane for six hours (lanes 2 to 3) induces caspase-3 cleavage (activation) as compared to control condition (lane 1). 2-APB (5 mg/kg: lanes 6 to 8; and 10 mg/kg: lanes 4 to 5) attenuates the sevoflurane-induced caspase-3 activation. B . Quantification of the Western blot shows that the sevoflurane anesthesia (black bar) induces caspase-3 activation compared with control condition (white bar). 2-APB (5 mg/kg: gray bar; and 10 mg/kg: net bar) attenuates the sevoflurane-induced caspase-3 activation (black bar). C . The sevoflurane anesthesia (lanes 3 to 4) increases Aβ levels as compared to control condition (lanes 1 to 2). The 2-APB treatment (lanes 5 to 6) attenuates the sevoflurane-induced increases of Aβ levels (lanes 3 to 4). D . Quantification of the Western blot shows that the sevoflurane anesthesia (black bar) increases Aβ levels compared with control condition (white bar). The 2-APB treatment (10 mg/kg, net bar) attenuates the sevoflurane-induced increase of Aβ (black bar). We have averaged results from four independent experiments. Aβ, β-amyloid protein; FL, full length. * or # P

    Article Snippet: The six-day-old neonatal naïve mice were treated with 3% sevoflurane plus 60% oxygen for six hours, the brain tissues were harvested at the end of the experiment and were subjected to Western blot analysis by which caspase-3 antibody was used to detect both caspase-3 fragment (17 – 20 kDa) and FL-caspase-3 (35 - 40 kDa).

    Techniques: Activation Assay, Mouse Assay, Western Blot

    Anesthesia with 3% sevoflurane for two hours does not induce caspase-3 activation in the brain tissues of neonatal naïve mice A . The anesthesia with 3% sevoflurane for two hours (lanes 3 to 5) does not induce caspase-3 cleavage (activation) as compared to control condition (lanes 1 to 2) in the brain tissues of neonatal naïve mice. B . Quantification of the Western blot shows that the anesthesia with 3% sevoflurane for two hours (black bar) does not induce caspase-3 activation compared with the control condition (white bar). We have averaged results from four independent experiments. FL, full length.

    Journal: Anesthesiology

    Article Title: Anesthetic Sevoflurane Causes Neurotoxicity Differently in Neonatal Na?ve and Alzheimer's Disease Transgenic Mice

    doi: 10.1097/ALN.0b013e3181d94de1

    Figure Lengend Snippet: Anesthesia with 3% sevoflurane for two hours does not induce caspase-3 activation in the brain tissues of neonatal naïve mice A . The anesthesia with 3% sevoflurane for two hours (lanes 3 to 5) does not induce caspase-3 cleavage (activation) as compared to control condition (lanes 1 to 2) in the brain tissues of neonatal naïve mice. B . Quantification of the Western blot shows that the anesthesia with 3% sevoflurane for two hours (black bar) does not induce caspase-3 activation compared with the control condition (white bar). We have averaged results from four independent experiments. FL, full length.

    Article Snippet: The six-day-old neonatal naïve mice were treated with 3% sevoflurane plus 60% oxygen for six hours, the brain tissues were harvested at the end of the experiment and were subjected to Western blot analysis by which caspase-3 antibody was used to detect both caspase-3 fragment (17 – 20 kDa) and FL-caspase-3 (35 - 40 kDa).

    Techniques: Activation Assay, Mouse Assay, Western Blot

    Anesthesia with 2.1% sevoflurane for six hours induces caspase-3 activation in the brain tissues of neonatal naïve and AD transgenic mice A . The anesthesia with 2.1% sevoflurane for six hours (lanes 4 to 6) induces caspase-3 cleavage (activation) as compared to control condition (lanes 1 to 3) in the brain tissues of neonatal naïve mice. B . Caspase-3 activation assessed by quantifying the ratio of cleaved (activated) caspase-3 fragment (17 – 20 kDa) to FL-caspase-3 (35 - 40 kDa) in the Western blot. Quantification of the Western blot shows that the sevoflurane anesthesia (black bar) induces caspase-3 activation compared with control condition (white bar). C . The anesthesia with 2.1% sevoflurane for six hours (lanes 4 to 6) induces caspase-3 cleavage (activation) as compared to control condition (lanes 1 to 3) in the brain tissues of neonatal AD transgenic mice. D . Quantification of the Western blot shows that the sevoflurane anesthesia (black bar) induces caspase-3 activation compared with control condition (white bar), normalized to β-Actin levels. We have averaged results from ten independent experiments. AD, Alzheimer's disease, FL, full length. * P

    Journal: Anesthesiology

    Article Title: Anesthetic Sevoflurane Causes Neurotoxicity Differently in Neonatal Na?ve and Alzheimer's Disease Transgenic Mice

    doi: 10.1097/ALN.0b013e3181d94de1

    Figure Lengend Snippet: Anesthesia with 2.1% sevoflurane for six hours induces caspase-3 activation in the brain tissues of neonatal naïve and AD transgenic mice A . The anesthesia with 2.1% sevoflurane for six hours (lanes 4 to 6) induces caspase-3 cleavage (activation) as compared to control condition (lanes 1 to 3) in the brain tissues of neonatal naïve mice. B . Caspase-3 activation assessed by quantifying the ratio of cleaved (activated) caspase-3 fragment (17 – 20 kDa) to FL-caspase-3 (35 - 40 kDa) in the Western blot. Quantification of the Western blot shows that the sevoflurane anesthesia (black bar) induces caspase-3 activation compared with control condition (white bar). C . The anesthesia with 2.1% sevoflurane for six hours (lanes 4 to 6) induces caspase-3 cleavage (activation) as compared to control condition (lanes 1 to 3) in the brain tissues of neonatal AD transgenic mice. D . Quantification of the Western blot shows that the sevoflurane anesthesia (black bar) induces caspase-3 activation compared with control condition (white bar), normalized to β-Actin levels. We have averaged results from ten independent experiments. AD, Alzheimer's disease, FL, full length. * P

    Article Snippet: The six-day-old neonatal naïve mice were treated with 3% sevoflurane plus 60% oxygen for six hours, the brain tissues were harvested at the end of the experiment and were subjected to Western blot analysis by which caspase-3 antibody was used to detect both caspase-3 fragment (17 – 20 kDa) and FL-caspase-3 (35 - 40 kDa).

    Techniques: Activation Assay, Transgenic Assay, Mouse Assay, Western Blot

    Anesthesia with 3% sevoflurane for six hours induces a greater degree of caspase-3 activation in the brain tissues of neonatal AD transgenic mice than that in neonatal naïve mice A . Anesthesia with the sevoflurane anesthesia (lanes 2 and 4) induces caspase-3 activation as compared to control condition (lanes 1 and 3) in the naïve mice and AD transgenic mice, respectively. The sevoflurane anesthesia induces a greater degree of caspase-3 activation in the AD transgenic mice (lane 4) than in naïve mice (lane 2). B . Quantification of the Western blot shows that the sevoflurane anesthesia (black bar and net bar) induces caspase-3 activation compared with control condition (white bar and gray bar) in both neonatal naïve and AD transgenic mice, respectively. The sevoflurane anesthesia induces a greater degree of caspase activation in the AD transgenic mice (net bar) than in the naïve mice (black bar). We have averaged results from four independent experiments. AD, Alzheimer's disease, FL, full length. ** or ## P

    Journal: Anesthesiology

    Article Title: Anesthetic Sevoflurane Causes Neurotoxicity Differently in Neonatal Na?ve and Alzheimer's Disease Transgenic Mice

    doi: 10.1097/ALN.0b013e3181d94de1

    Figure Lengend Snippet: Anesthesia with 3% sevoflurane for six hours induces a greater degree of caspase-3 activation in the brain tissues of neonatal AD transgenic mice than that in neonatal naïve mice A . Anesthesia with the sevoflurane anesthesia (lanes 2 and 4) induces caspase-3 activation as compared to control condition (lanes 1 and 3) in the naïve mice and AD transgenic mice, respectively. The sevoflurane anesthesia induces a greater degree of caspase-3 activation in the AD transgenic mice (lane 4) than in naïve mice (lane 2). B . Quantification of the Western blot shows that the sevoflurane anesthesia (black bar and net bar) induces caspase-3 activation compared with control condition (white bar and gray bar) in both neonatal naïve and AD transgenic mice, respectively. The sevoflurane anesthesia induces a greater degree of caspase activation in the AD transgenic mice (net bar) than in the naïve mice (black bar). We have averaged results from four independent experiments. AD, Alzheimer's disease, FL, full length. ** or ## P

    Article Snippet: The six-day-old neonatal naïve mice were treated with 3% sevoflurane plus 60% oxygen for six hours, the brain tissues were harvested at the end of the experiment and were subjected to Western blot analysis by which caspase-3 antibody was used to detect both caspase-3 fragment (17 – 20 kDa) and FL-caspase-3 (35 - 40 kDa).

    Techniques: Activation Assay, Transgenic Assay, Mouse Assay, Western Blot

    Anesthesia with 3% sevoflurane for six hours induces caspase-3 activation and APP processing in the brain tissues of neonatal naïve mice A . The anesthesia with 3% sevoflurane for six hours (lanes 5 to 8) induces caspase-3 cleavage (activation) as compared to control condition (lanes 1 to 4) in the brain tissues of neonatal naïve mice. B . Caspase-3 activation assessed by quantifying the ratio of cleaved (activated) caspase-3 fragment (17–20 kDa) to FL-caspase-3 (35 - 40 kDa) in the Western blot. Quantification of the Western blot shows that the sevoflurane anesthesia (black bar) induces caspase-3 activation compared with control condition (white bar). C . The sevoflurane anesthesia (lanes 4 to 6) reduces levels of APP-C83 and APP-C99 as compared to control condition (lanes 1 to 3). D . Quantification of the Western blot shows that the sevoflurane anesthesia (black bar) decreases the ratio of APP-C83 to APP-FL as compared to control condition (white bar). E . Quantification of the Western blot shows that the sevoflurane anesthesia (black bar) decreases the ratio of APP-C99 to APP-FL as compared to control condition (white bar). We have averaged results from six independent experiments. APP, amyloid precursor protein; FL, full length. * P

    Journal: Anesthesiology

    Article Title: Anesthetic Sevoflurane Causes Neurotoxicity Differently in Neonatal Na?ve and Alzheimer's Disease Transgenic Mice

    doi: 10.1097/ALN.0b013e3181d94de1

    Figure Lengend Snippet: Anesthesia with 3% sevoflurane for six hours induces caspase-3 activation and APP processing in the brain tissues of neonatal naïve mice A . The anesthesia with 3% sevoflurane for six hours (lanes 5 to 8) induces caspase-3 cleavage (activation) as compared to control condition (lanes 1 to 4) in the brain tissues of neonatal naïve mice. B . Caspase-3 activation assessed by quantifying the ratio of cleaved (activated) caspase-3 fragment (17–20 kDa) to FL-caspase-3 (35 - 40 kDa) in the Western blot. Quantification of the Western blot shows that the sevoflurane anesthesia (black bar) induces caspase-3 activation compared with control condition (white bar). C . The sevoflurane anesthesia (lanes 4 to 6) reduces levels of APP-C83 and APP-C99 as compared to control condition (lanes 1 to 3). D . Quantification of the Western blot shows that the sevoflurane anesthesia (black bar) decreases the ratio of APP-C83 to APP-FL as compared to control condition (white bar). E . Quantification of the Western blot shows that the sevoflurane anesthesia (black bar) decreases the ratio of APP-C99 to APP-FL as compared to control condition (white bar). We have averaged results from six independent experiments. APP, amyloid precursor protein; FL, full length. * P

    Article Snippet: The six-day-old neonatal naïve mice were treated with 3% sevoflurane plus 60% oxygen for six hours, the brain tissues were harvested at the end of the experiment and were subjected to Western blot analysis by which caspase-3 antibody was used to detect both caspase-3 fragment (17 – 20 kDa) and FL-caspase-3 (35 - 40 kDa).

    Techniques: Activation Assay, Mouse Assay, Western Blot

    Antioxidant treatment promotes the survival of cochlear HCs after neomycin injury. (A) Immunofluorescence staining with Mito-SOX and MYO7A in the middle turn of the cochlea after different treatments, n = 4. (B) Quantification of the numbers of Mito-SOX and MYO7A double-positive cells in (A). (C) Quantification of the MYO7A-positive cells in (A). (D) Immunofluorescence staining for cleaved-CASP3 and MYO7A in the middle turn of the cochlea after different treatments, n = 4. (E) Immunofluorescence staining for TUNEL and MYO7A in the middle turn of the cochlea after different treatments, n = 4. (F and G) Quantification of the numbers of positive cells in (Dand E). The neomycin-induced oxidative stress and apoptosis in HCs were significantly reduced after pretreatment with NAC. Scale bars: 20 μm. * P

    Journal: Autophagy

    Article Title: Autophagy protects auditory hair cells against neomycin-induced damage

    doi: 10.1080/15548627.2017.1359449

    Figure Lengend Snippet: Antioxidant treatment promotes the survival of cochlear HCs after neomycin injury. (A) Immunofluorescence staining with Mito-SOX and MYO7A in the middle turn of the cochlea after different treatments, n = 4. (B) Quantification of the numbers of Mito-SOX and MYO7A double-positive cells in (A). (C) Quantification of the MYO7A-positive cells in (A). (D) Immunofluorescence staining for cleaved-CASP3 and MYO7A in the middle turn of the cochlea after different treatments, n = 4. (E) Immunofluorescence staining for TUNEL and MYO7A in the middle turn of the cochlea after different treatments, n = 4. (F and G) Quantification of the numbers of positive cells in (Dand E). The neomycin-induced oxidative stress and apoptosis in HCs were significantly reduced after pretreatment with NAC. Scale bars: 20 μm. * P

    Article Snippet: Anti-cleaved-CASP3 antibody (Cell Signaling Technology, 9664S), anti-cleaved-PARP1 antibody (Cell Signaling Technology, 9544S), Mito-SOX Red (Life Technologies, M36008), anti-LC3B antibody (Santa Cruz Biotechnology, sc-16755), anti-SQSTM1/p62 antibody (Abcam, ab109012), anti-ATG5 antibody (Cell Signaling Technology, 12994), anti-BECN1 antibody (Cell Signaling Technology, 3495), anti-ATG7 antibody (Cell Signaling Technology, 8558S), anti-MYO7A antibody (Proteus Bioscience, 25–6790), anti-TOMM20 antibody (Proteintech, 11802–1-AP), and DAPI (Sigma-Aldrich, D9542) were used to analyze apoptotic cells, detect autophagy, measure ROS, stain HCs, measure mitochondrial number, and stain nuclei, respectively.

    Techniques: Immunofluorescence, Staining, TUNEL Assay

    Autophagy affects cell death and apoptosis in HEI-OC-1 cells after neomycin exposure. (A) Apoptosis analysis by flow cytometry after different treatments. The lower left quadrants, lower right quadrants, and upper right quadrants of the images represent live cells, early apoptotic cells, and late apoptotic cells, respectively, n = 4. (B) Quantification of the flow cytometry data. The proportions of dead cells and early apoptotic cells after neomycin treatment were significantly increased compared with the undamaged groups. In addition, the dead and apoptotic proportions could be increased by 3-MA and ATG5 knockdown, and the apoptotic proportions could be reduced by rapamycin. (C) TUNEL and DAPI double staining showing the apoptotic HEI-OC-1 cells after different treatments, n = 4. (D) Cleaved-CASP3 and DAPI double staining confirmed the apoptotic cells after different treatments, n = 4. (E and F) Quantification of the numbers of TUNEL and DAPI double-positive, as well as cleaved-CASP3 and DAPI double-positive cells in (C and D), respectively.

    Journal: Autophagy

    Article Title: Autophagy protects auditory hair cells against neomycin-induced damage

    doi: 10.1080/15548627.2017.1359449

    Figure Lengend Snippet: Autophagy affects cell death and apoptosis in HEI-OC-1 cells after neomycin exposure. (A) Apoptosis analysis by flow cytometry after different treatments. The lower left quadrants, lower right quadrants, and upper right quadrants of the images represent live cells, early apoptotic cells, and late apoptotic cells, respectively, n = 4. (B) Quantification of the flow cytometry data. The proportions of dead cells and early apoptotic cells after neomycin treatment were significantly increased compared with the undamaged groups. In addition, the dead and apoptotic proportions could be increased by 3-MA and ATG5 knockdown, and the apoptotic proportions could be reduced by rapamycin. (C) TUNEL and DAPI double staining showing the apoptotic HEI-OC-1 cells after different treatments, n = 4. (D) Cleaved-CASP3 and DAPI double staining confirmed the apoptotic cells after different treatments, n = 4. (E and F) Quantification of the numbers of TUNEL and DAPI double-positive, as well as cleaved-CASP3 and DAPI double-positive cells in (C and D), respectively.

    Article Snippet: Anti-cleaved-CASP3 antibody (Cell Signaling Technology, 9664S), anti-cleaved-PARP1 antibody (Cell Signaling Technology, 9544S), Mito-SOX Red (Life Technologies, M36008), anti-LC3B antibody (Santa Cruz Biotechnology, sc-16755), anti-SQSTM1/p62 antibody (Abcam, ab109012), anti-ATG5 antibody (Cell Signaling Technology, 12994), anti-BECN1 antibody (Cell Signaling Technology, 3495), anti-ATG7 antibody (Cell Signaling Technology, 8558S), anti-MYO7A antibody (Proteus Bioscience, 25–6790), anti-TOMM20 antibody (Proteintech, 11802–1-AP), and DAPI (Sigma-Aldrich, D9542) were used to analyze apoptotic cells, detect autophagy, measure ROS, stain HCs, measure mitochondrial number, and stain nuclei, respectively.

    Techniques: Flow Cytometry, Cytometry, TUNEL Assay, Double Staining

    Antioxidant treatment promotes the survival of HEI-OC-1 cells after neomycin injury. (A) TUNEL and DAPI double staining and (B) cleaved-CASP3 and DAPI double staining showed the number of apoptotic HEI-OC-1 cells after the different treatments, n = 4. (C) Quantification of the numbers of TUNEL and DAPI double-positive, as well as cleaved-CASP3 and DAPI double-positive cells in (A and B), respectively. The apoptotic cells were significantly decreased after pretreatment with NAC. For flow cytometry quantification experiments, the values for the normal controls were set to 1. For all experiments, * P

    Journal: Autophagy

    Article Title: Autophagy protects auditory hair cells against neomycin-induced damage

    doi: 10.1080/15548627.2017.1359449

    Figure Lengend Snippet: Antioxidant treatment promotes the survival of HEI-OC-1 cells after neomycin injury. (A) TUNEL and DAPI double staining and (B) cleaved-CASP3 and DAPI double staining showed the number of apoptotic HEI-OC-1 cells after the different treatments, n = 4. (C) Quantification of the numbers of TUNEL and DAPI double-positive, as well as cleaved-CASP3 and DAPI double-positive cells in (A and B), respectively. The apoptotic cells were significantly decreased after pretreatment with NAC. For flow cytometry quantification experiments, the values for the normal controls were set to 1. For all experiments, * P

    Article Snippet: Anti-cleaved-CASP3 antibody (Cell Signaling Technology, 9664S), anti-cleaved-PARP1 antibody (Cell Signaling Technology, 9544S), Mito-SOX Red (Life Technologies, M36008), anti-LC3B antibody (Santa Cruz Biotechnology, sc-16755), anti-SQSTM1/p62 antibody (Abcam, ab109012), anti-ATG5 antibody (Cell Signaling Technology, 12994), anti-BECN1 antibody (Cell Signaling Technology, 3495), anti-ATG7 antibody (Cell Signaling Technology, 8558S), anti-MYO7A antibody (Proteus Bioscience, 25–6790), anti-TOMM20 antibody (Proteintech, 11802–1-AP), and DAPI (Sigma-Aldrich, D9542) were used to analyze apoptotic cells, detect autophagy, measure ROS, stain HCs, measure mitochondrial number, and stain nuclei, respectively.

    Techniques: TUNEL Assay, Double Staining, Flow Cytometry, Cytometry

    Autophagy affects the levels of apoptosis-related genes and proteins in HEI-OC-1 cells after neomycin exposure. (A) Western blots with anti-cleaved-CASP3 antibody revealed that the amount of cleaved-CASP3 is significantly increased after neomycin treatment. In addition, the amount could be increased by ATG knockdown and could be reduced by rapamycin, n = 4. (B) Quantification of the western blot in (A). (C) Western blots with anti-cleaved-PARP1 antibody, n = 4. (D) Quantification of the western blot in (C). (E) The mRNA levels of proapoptotic genes and antiapoptotic genes were analyzed by qRT-PCR after treatment with neomycin, n = 3. (F) qRT-PCR analysis of the apoptosis-related gene expression in the ATG5 knockdown group, 3-MA-pretreatment group, and rapamycin-pretreatment group after neomycin injury, n = 3. For qRT-PCR experiments, the values for the normal controls were set to 1. Scale bars: 20 μm. * P

    Journal: Autophagy

    Article Title: Autophagy protects auditory hair cells against neomycin-induced damage

    doi: 10.1080/15548627.2017.1359449

    Figure Lengend Snippet: Autophagy affects the levels of apoptosis-related genes and proteins in HEI-OC-1 cells after neomycin exposure. (A) Western blots with anti-cleaved-CASP3 antibody revealed that the amount of cleaved-CASP3 is significantly increased after neomycin treatment. In addition, the amount could be increased by ATG knockdown and could be reduced by rapamycin, n = 4. (B) Quantification of the western blot in (A). (C) Western blots with anti-cleaved-PARP1 antibody, n = 4. (D) Quantification of the western blot in (C). (E) The mRNA levels of proapoptotic genes and antiapoptotic genes were analyzed by qRT-PCR after treatment with neomycin, n = 3. (F) qRT-PCR analysis of the apoptosis-related gene expression in the ATG5 knockdown group, 3-MA-pretreatment group, and rapamycin-pretreatment group after neomycin injury, n = 3. For qRT-PCR experiments, the values for the normal controls were set to 1. Scale bars: 20 μm. * P

    Article Snippet: Anti-cleaved-CASP3 antibody (Cell Signaling Technology, 9664S), anti-cleaved-PARP1 antibody (Cell Signaling Technology, 9544S), Mito-SOX Red (Life Technologies, M36008), anti-LC3B antibody (Santa Cruz Biotechnology, sc-16755), anti-SQSTM1/p62 antibody (Abcam, ab109012), anti-ATG5 antibody (Cell Signaling Technology, 12994), anti-BECN1 antibody (Cell Signaling Technology, 3495), anti-ATG7 antibody (Cell Signaling Technology, 8558S), anti-MYO7A antibody (Proteus Bioscience, 25–6790), anti-TOMM20 antibody (Proteintech, 11802–1-AP), and DAPI (Sigma-Aldrich, D9542) were used to analyze apoptotic cells, detect autophagy, measure ROS, stain HCs, measure mitochondrial number, and stain nuclei, respectively.

    Techniques: Western Blot, Quantitative RT-PCR, Expressing

    Autophagy modulates the injury severity in cochlear HCs after neomycin exposure. (A) Immunofluorescence staining with TUNEL and MYO7A in the middle turn of the cochlea after different treatments, n = 3. (B) Immunofluorescence staining for cleaved-CASP3 and MYO7A in the middle turn of the cochlea after different treatments. (C and D) Quantification of the numbers of TUNEL and MYO7A double-positive, as well as cleaved-CASP3 and MYO7A double-positive cells. The numbers and proportions of cleaved CASP3-positive cells and TUNEL-positive cells in the neomycin-treated groups were significantly greater than the undamaged controls. Moreover, the numbers of apoptotic cells were significantly increased by 3-MA and decreased by rapamycin, n = 3. (E) The mRNA levels of 8 apoptosis-related genes were analyzed by qRT-PCR after treatment with neomycin, n = 4. (F) qRT-PCR analysis of the apoptosis-related gene expression in the 3-MA-pretreatment group and rapamycin-pretreatment group after neomycin injury, n = 4. For qRT-PCR experiments, the values for the normal controls were set to 1. Scale bars: 20 μm. * P

    Journal: Autophagy

    Article Title: Autophagy protects auditory hair cells against neomycin-induced damage

    doi: 10.1080/15548627.2017.1359449

    Figure Lengend Snippet: Autophagy modulates the injury severity in cochlear HCs after neomycin exposure. (A) Immunofluorescence staining with TUNEL and MYO7A in the middle turn of the cochlea after different treatments, n = 3. (B) Immunofluorescence staining for cleaved-CASP3 and MYO7A in the middle turn of the cochlea after different treatments. (C and D) Quantification of the numbers of TUNEL and MYO7A double-positive, as well as cleaved-CASP3 and MYO7A double-positive cells. The numbers and proportions of cleaved CASP3-positive cells and TUNEL-positive cells in the neomycin-treated groups were significantly greater than the undamaged controls. Moreover, the numbers of apoptotic cells were significantly increased by 3-MA and decreased by rapamycin, n = 3. (E) The mRNA levels of 8 apoptosis-related genes were analyzed by qRT-PCR after treatment with neomycin, n = 4. (F) qRT-PCR analysis of the apoptosis-related gene expression in the 3-MA-pretreatment group and rapamycin-pretreatment group after neomycin injury, n = 4. For qRT-PCR experiments, the values for the normal controls were set to 1. Scale bars: 20 μm. * P

    Article Snippet: Anti-cleaved-CASP3 antibody (Cell Signaling Technology, 9664S), anti-cleaved-PARP1 antibody (Cell Signaling Technology, 9544S), Mito-SOX Red (Life Technologies, M36008), anti-LC3B antibody (Santa Cruz Biotechnology, sc-16755), anti-SQSTM1/p62 antibody (Abcam, ab109012), anti-ATG5 antibody (Cell Signaling Technology, 12994), anti-BECN1 antibody (Cell Signaling Technology, 3495), anti-ATG7 antibody (Cell Signaling Technology, 8558S), anti-MYO7A antibody (Proteus Bioscience, 25–6790), anti-TOMM20 antibody (Proteintech, 11802–1-AP), and DAPI (Sigma-Aldrich, D9542) were used to analyze apoptotic cells, detect autophagy, measure ROS, stain HCs, measure mitochondrial number, and stain nuclei, respectively.

    Techniques: Immunofluorescence, Staining, TUNEL Assay, Quantitative RT-PCR, Expressing

    Activity of ABT-737 against SCLC primary xenografts. A, lysates from LX22, LX33, and LX36 primary xenografts probed with Bcl-2, Bcl-x L , Mcl-1, Noxa, and Actin; lysates from the H345 SCLC cell line used as a reference. B, caspase-3 activation in an LX22

    Journal:

    Article Title: Therapeutic Efficacy of ABT-737, a Selective Inhibitor of BCL-2, in Small Cell Lung Cancer

    doi: 10.1158/0008-5472.CAN-07-5031

    Figure Lengend Snippet: Activity of ABT-737 against SCLC primary xenografts. A, lysates from LX22, LX33, and LX36 primary xenografts probed with Bcl-2, Bcl-x L , Mcl-1, Noxa, and Actin; lysates from the H345 SCLC cell line used as a reference. B, caspase-3 activation in an LX22

    Article Snippet: The sections were prepared as described above and incubated with anti-activated caspase-3 antibody (Cell Signaling Technologies) and visualized using an horseradish peroxidase–conjugated secondary antibody and 3,3′-diaminobenzidine reagent (Dako Envision+, Dako).

    Techniques: Activity Assay, Activation Assay

    LM-γ2 functional significance. Inhibition of LM-γ2 ( A ) reduced cell invasion ( B ) and Src activity ( C ). ( D ) LM-γ2 induces resistance to apoptosis, as evidenced by a decrease in Caspase-3 levels in control cells when compared with

    Journal: Human Molecular Genetics

    Article Title: E-cadherin-defective gastric cancer cells depend on Laminin to survive and invade

    doi: 10.1093/hmg/ddv312

    Figure Lengend Snippet: LM-γ2 functional significance. Inhibition of LM-γ2 ( A ) reduced cell invasion ( B ) and Src activity ( C ). ( D ) LM-γ2 induces resistance to apoptosis, as evidenced by a decrease in Caspase-3 levels in control cells when compared with

    Article Snippet: Primary antibodies included mouse anti-Ecad (1:1000, BD Biosciences, San Jose, CA, USA), mouse anti-α-tubulin (1:10 000, Sigma), mouse anti-laminin γ2, clone D4B5 (1:250, Merck Millipore, Darmstadt, Germany), mouse anti-laminin α3, clone 12C4 (1:500, Merck Millipore), mouse anti-integrin β1 (1:1000, BD Biosciences), rabbit anti-integrin β4 (1:1000, Cell Signalling), rabbit anti-Phospho SAPK/JNK(Thr183/Tyr185) (1:1000, Cell Signalling), rabbit anti-SAPK/JNK (1:1000, Cell Signalling), rabbit anti-Phospho Akt(Thr308) (1:1000, Cell Signalling), rabbit anti-Phospho Src(Tyr416) (1:1000, Cell Signalling) and mouse anti-Casp3 (1:1000, Cell Signalling).

    Techniques: Functional Assay, Inhibition, Activity Assay

    Effect of lidocaine on hypoxia-induced apoptosis of H9c2 cells. Cell apoptosis was measured using a FITC-Annexin V/PI detection kit. (A) Representative images of flow cytometry data and (B) apoptosis rates. (C) Representative images of western blotting analysis of Bax, Bcl-2 and caspase-3. **P

    Journal: Experimental and Therapeutic Medicine

    Article Title: Lidocaine protects H9c2 cells from hypoxia-induced injury through regulation of the MAPK/ERK/NF-κB signaling pathway

    doi: 10.3892/etm.2019.8055

    Figure Lengend Snippet: Effect of lidocaine on hypoxia-induced apoptosis of H9c2 cells. Cell apoptosis was measured using a FITC-Annexin V/PI detection kit. (A) Representative images of flow cytometry data and (B) apoptosis rates. (C) Representative images of western blotting analysis of Bax, Bcl-2 and caspase-3. **P

    Article Snippet: The membrane was blocked with 5% skim milk for 1 h at room temperature and then probed with primary antibodies: Bcl-2 (cat no. ab196495; 1:1,000; Abcam), Bax (cat no. 14796; 1:1,000; Cell Signaling Technology, Inc.), caspase-3 (cat no. 14220; 1:1,000; Cell Signaling Technology, Inc.), NF-κB p65 (cat no. 8242; 1:1,000; Cell Signaling Technology, Inc.), phosphorylated (p)-p65 (cat no. 3033; 1:1,000; Cell Signaling Technology, Inc.), ERK1/2 (cat no. 4695; 1:1,000; Cell Signaling Technology, Inc.), p-ERK1/2 (cat no. 4376; 1:1,000; Cell Signaling Technology, Inc.) and β-actin (cat no. 4970; 1:1,000; Cell Signaling Technology, Inc.), at 4°C overnight.

    Techniques: Flow Cytometry, Cytometry, Western Blot

    DUSP6 SUMOylation at K234 is protective to brain neurons in ischemic stroke. ( A ) Brain ischemia and brain damage in the tMCAO model. Left: Regional cerebral blood flow in the core of the middle cerebral artery territory of C57BL/6 mice before, during, and after ischemia as measured by LDF. Flow values were averaged at 5-min intervals and expressed as means ± SEM of nine experiments. Right: Representative photographs of TTC-stained brain sections (1 mm) showing brain infarction at 24 hours after the onset of ischemia. ( B ) Time-dependent decreases in DUSP6 SUMOylation and DUSP6 protein levels during reperfusion in mice subjected to tMCAO for 40 min. Cerebral cortices from C57BL/6 mice (wild type) that underwent 40 min tMCAO, followed by different periods of reperfusion as indicated, were lysed under denaturing conditions and subjected to IP with anti-DUSP6 (or the IgG control), which was followed by IB using anti-SUMO1 and anti-DUSP6. β-Actin was used as the loading control. Note also the time-dependent decrease in overall protein SUMOylation. ( C to G ) Transcriptional up-regulation of SENP1 during reperfusion of tMCAO injured mice. The original lysates as in (B) were analyzed by real-time quantitative RT-PCR for mRNA levels of DUSP6 (C), SAE1/SAE2 (D), UBC9 (E), and SENP1 (F), as well as by IB for protein levels of SAE1, SAE2, UBC9, and SENP1, with β-actin as the loading control (G). Only SENP1 showed time-dependent increases at both the mRNA (F) and protein levels (G). ( H ) Schematic representation of adeno-associated virus (AAV) infection into mouse primary somatosensory cortex, barrel field, and secondary somatosensory cortex (left) and representative brain images showing the expression of mNeonGreen in cerebral cortex (right). The boxed area is enhanced by high magnification to show mNeonGreen-positive neurons. Scale bar, 100 μm. ( I to K ) Overexpression of wild-type DUSP6 reduced, but the SUMOylation-deficient DUSP6 K234R mutant exacerbated, cortical neuron apoptosis in response to brain I/R. AAV-DUSP6, AAV-DUSP6 K234R , or a control AAV driven by the human synapsin promoter (hSyn) was injected into the primary somatosensory cortex, barrel field, and secondary somatosensory cortex of adult wild-type C57BL/6 mice as shown in (H). The animals were subjected to 40-min tMCAO, followed by 24-hour reperfusion at 4 weeks after stereotactic intracerebral injection. (I) The cerebral cortical tissues were lysed and subjected to IB using anti-cleaved caspase-3, anti–p–Drp1-S616, anti–p–Drp1-S637, anti-Drp1, anti-Flag, and anti-DUSP6. β-Actin was used as the loading control. Caspase-3 cleavage was reduced by AAV-DUSP6 but increased by AAV-DUSP6 K234R . Note also the corresponding changes in p–Drp1-S616 levels; however, p–Drp1-S637 levels were affected by neither AAV-DUSP6 nor AAV-DUSP6 K234R , despite the decrease caused by I/R. ( J ) The tissues were sectioned and stained by TUNEL (red) and DAPI (blue). The occurrence of TUNEL + cells in areas with DUSP6 expression (green) was significantly less than that with DUSP6 K234R expression. Representative confocal images in virus-infected cortical areas of brain sections are shown at the left. Scale bar, 100 μm. Quantification of TUNEL + cells are shown at the right. Data represent means ± SEM of n = 3 independent experiments, with six to eight fields of each group used for statistics in each experiment. * P

    Journal: Science Advances

    Article Title: DUSP6 SUMOylation protects cells from oxidative damage via direct regulation of Drp1 dephosphorylation

    doi: 10.1126/sciadv.aaz0361

    Figure Lengend Snippet: DUSP6 SUMOylation at K234 is protective to brain neurons in ischemic stroke. ( A ) Brain ischemia and brain damage in the tMCAO model. Left: Regional cerebral blood flow in the core of the middle cerebral artery territory of C57BL/6 mice before, during, and after ischemia as measured by LDF. Flow values were averaged at 5-min intervals and expressed as means ± SEM of nine experiments. Right: Representative photographs of TTC-stained brain sections (1 mm) showing brain infarction at 24 hours after the onset of ischemia. ( B ) Time-dependent decreases in DUSP6 SUMOylation and DUSP6 protein levels during reperfusion in mice subjected to tMCAO for 40 min. Cerebral cortices from C57BL/6 mice (wild type) that underwent 40 min tMCAO, followed by different periods of reperfusion as indicated, were lysed under denaturing conditions and subjected to IP with anti-DUSP6 (or the IgG control), which was followed by IB using anti-SUMO1 and anti-DUSP6. β-Actin was used as the loading control. Note also the time-dependent decrease in overall protein SUMOylation. ( C to G ) Transcriptional up-regulation of SENP1 during reperfusion of tMCAO injured mice. The original lysates as in (B) were analyzed by real-time quantitative RT-PCR for mRNA levels of DUSP6 (C), SAE1/SAE2 (D), UBC9 (E), and SENP1 (F), as well as by IB for protein levels of SAE1, SAE2, UBC9, and SENP1, with β-actin as the loading control (G). Only SENP1 showed time-dependent increases at both the mRNA (F) and protein levels (G). ( H ) Schematic representation of adeno-associated virus (AAV) infection into mouse primary somatosensory cortex, barrel field, and secondary somatosensory cortex (left) and representative brain images showing the expression of mNeonGreen in cerebral cortex (right). The boxed area is enhanced by high magnification to show mNeonGreen-positive neurons. Scale bar, 100 μm. ( I to K ) Overexpression of wild-type DUSP6 reduced, but the SUMOylation-deficient DUSP6 K234R mutant exacerbated, cortical neuron apoptosis in response to brain I/R. AAV-DUSP6, AAV-DUSP6 K234R , or a control AAV driven by the human synapsin promoter (hSyn) was injected into the primary somatosensory cortex, barrel field, and secondary somatosensory cortex of adult wild-type C57BL/6 mice as shown in (H). The animals were subjected to 40-min tMCAO, followed by 24-hour reperfusion at 4 weeks after stereotactic intracerebral injection. (I) The cerebral cortical tissues were lysed and subjected to IB using anti-cleaved caspase-3, anti–p–Drp1-S616, anti–p–Drp1-S637, anti-Drp1, anti-Flag, and anti-DUSP6. β-Actin was used as the loading control. Caspase-3 cleavage was reduced by AAV-DUSP6 but increased by AAV-DUSP6 K234R . Note also the corresponding changes in p–Drp1-S616 levels; however, p–Drp1-S637 levels were affected by neither AAV-DUSP6 nor AAV-DUSP6 K234R , despite the decrease caused by I/R. ( J ) The tissues were sectioned and stained by TUNEL (red) and DAPI (blue). The occurrence of TUNEL + cells in areas with DUSP6 expression (green) was significantly less than that with DUSP6 K234R expression. Representative confocal images in virus-infected cortical areas of brain sections are shown at the left. Scale bar, 100 μm. Quantification of TUNEL + cells are shown at the right. Data represent means ± SEM of n = 3 independent experiments, with six to eight fields of each group used for statistics in each experiment. * P

    Article Snippet: Antibodies and reagents Anti-cleaved caspase-3 (1:1000; 9661), anti-ERK1/2 (1:1000; 9102), anti–p-ERK1/2 (1:1000; 9106), anti-SAE1 (1:1000; 8688), anti–p–Drp1-S616 (1:1000; 3455), and anti–p–Drp1-S637 (1:1000; 4867) were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Mouse Assay, Staining, Quantitative RT-PCR, Infection, Expressing, Over Expression, Mutagenesis, Injection, TUNEL Assay

    Increasing DUSP6 SUMOylation suppresses oxidation-induced mitochondrial fragmentation and cell apoptosis. ( A and B ) H 2 O 2 treatment increased SENP1 levels in a concentration- and time-dependent manner. Lysates from untransfected HeLa cells treated with H 2 O 2 at different concentrations for 1 hour (A) and at 0.5 mM for different time periods (B) as indicated were analyzed by IB for levels of SENP1, SUMO1, and DUSP6, with β-actin used as the loading control. Note that the increases in SENP1 were accompanied with decreases in overall protein SUMOylation and endogenous DUSP6 levels. ( C ) H 2 O 2 -induced increase in caspase-3 cleavage is accompanied with an increase in SENP1 expression. Lysates from untransfected HeLa cells untreated or treated with 0.5 mM H 2 O 2 for 1 hour were analyzed as in (A) and (B), with an additional analysis for cleaved caspase-3. ( D ) Overexpression of DUSP6 suppressed H 2 O 2 -induced caspase-3 cleavage in a SUMOylation-dependent manner. HeLa cells transiently cotransfected with an empty vector (−), a vector encoding Flag-DUSP6 (wild type) or the Flag-DUSP6 K234R mutant, and HA-SUMO1 as indicated were untreated or treated with 0.5 mM H 2 O 2 for 1 hour beginning at 24 hours after transfection. Cell lysates were analyzed by IB for levels of cleaved caspase-3, Flag-DUSP6, p-ERK1/2, and ERK1/2, with β-actin used as the loading control. The overexpression of Flag-DUSP6 decreased H 2 O 2 -induced caspase-3 cleavage and the p-ERK1/2 levels, both of which were further suppressed by HA-SUMO1; however, Flag-DUSP6 K234R did not have these effects. In (A) to (D), blots are representatives of at least three independent experiments. ( E and F ) Overexpression of DUSP6 attenuated H 2 O 2 -induced cell apoptosis (E) and mitochondrial fragmentation (F) in a SUMOylation-dependent manner. H 2 O 2 -treated cells as described in (D) were either fixed and then stained for IF labeling of Flag for Flag-DUSP6 expression (green) and cell apoptosis (red) by TUNEL assay system (E) or incubated with MitoTracker Red (red) for 50 min at 37°C before fixation and IF staining for Flag (F). DAPI (blue) was used as the nuclear counterstaining. Representative confocal images are shown at the left (E ) or at the top (F ) . Scale bars, 100 μm (E) and 10 μm (F). Black and white images in (F) show magnification of boxed areas for MitoTracker. Quantification data represent means ± SEM of n = 3 independent experiments. * P

    Journal: Science Advances

    Article Title: DUSP6 SUMOylation protects cells from oxidative damage via direct regulation of Drp1 dephosphorylation

    doi: 10.1126/sciadv.aaz0361

    Figure Lengend Snippet: Increasing DUSP6 SUMOylation suppresses oxidation-induced mitochondrial fragmentation and cell apoptosis. ( A and B ) H 2 O 2 treatment increased SENP1 levels in a concentration- and time-dependent manner. Lysates from untransfected HeLa cells treated with H 2 O 2 at different concentrations for 1 hour (A) and at 0.5 mM for different time periods (B) as indicated were analyzed by IB for levels of SENP1, SUMO1, and DUSP6, with β-actin used as the loading control. Note that the increases in SENP1 were accompanied with decreases in overall protein SUMOylation and endogenous DUSP6 levels. ( C ) H 2 O 2 -induced increase in caspase-3 cleavage is accompanied with an increase in SENP1 expression. Lysates from untransfected HeLa cells untreated or treated with 0.5 mM H 2 O 2 for 1 hour were analyzed as in (A) and (B), with an additional analysis for cleaved caspase-3. ( D ) Overexpression of DUSP6 suppressed H 2 O 2 -induced caspase-3 cleavage in a SUMOylation-dependent manner. HeLa cells transiently cotransfected with an empty vector (−), a vector encoding Flag-DUSP6 (wild type) or the Flag-DUSP6 K234R mutant, and HA-SUMO1 as indicated were untreated or treated with 0.5 mM H 2 O 2 for 1 hour beginning at 24 hours after transfection. Cell lysates were analyzed by IB for levels of cleaved caspase-3, Flag-DUSP6, p-ERK1/2, and ERK1/2, with β-actin used as the loading control. The overexpression of Flag-DUSP6 decreased H 2 O 2 -induced caspase-3 cleavage and the p-ERK1/2 levels, both of which were further suppressed by HA-SUMO1; however, Flag-DUSP6 K234R did not have these effects. In (A) to (D), blots are representatives of at least three independent experiments. ( E and F ) Overexpression of DUSP6 attenuated H 2 O 2 -induced cell apoptosis (E) and mitochondrial fragmentation (F) in a SUMOylation-dependent manner. H 2 O 2 -treated cells as described in (D) were either fixed and then stained for IF labeling of Flag for Flag-DUSP6 expression (green) and cell apoptosis (red) by TUNEL assay system (E) or incubated with MitoTracker Red (red) for 50 min at 37°C before fixation and IF staining for Flag (F). DAPI (blue) was used as the nuclear counterstaining. Representative confocal images are shown at the left (E ) or at the top (F ) . Scale bars, 100 μm (E) and 10 μm (F). Black and white images in (F) show magnification of boxed areas for MitoTracker. Quantification data represent means ± SEM of n = 3 independent experiments. * P

    Article Snippet: Antibodies and reagents Anti-cleaved caspase-3 (1:1000; 9661), anti-ERK1/2 (1:1000; 9102), anti–p-ERK1/2 (1:1000; 9106), anti-SAE1 (1:1000; 8688), anti–p–Drp1-S616 (1:1000; 3455), and anti–p–Drp1-S637 (1:1000; 4867) were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Concentration Assay, Expressing, Over Expression, Plasmid Preparation, Mutagenesis, Transfection, Staining, Labeling, TUNEL Assay, Incubation

    H 2 O 2 destabilizes DUSP6 via ubiquitin-mediated degradation, and overexpression of DUSP6 protects cells against oxidative stress. ( A and B ) H 2 O 2 treatment decreased the levels of endogenous DUSP6 proteins in a dose- and time-dependent manner. Lysates from HeLa cells treated with H 2 O 2 at different concentrations for 1 hour (A) and at 0.5 mM for different times (B) as indicated were analyzed by immunoblotting (IB) using an anti-DUSP6 antibody. β-Actin was used as the loading control. ( C and D ) H 2 O 2 treatment increased caspase-3 cleavage (C) and DUSP6 ubiquitination (D). Lysates from HeLa cells untreated or treated with 0.5 mM H 2 O 2 for 1 hour were subjected to IB with anti-cleaved caspase-3 and anti-DUSP6 antibodies (C) or they were subjected to immunoprecipitation (IP) with the DUSP6 antibody [or control immunoglobulin G (IgG)] before being used for IB with anti-Ubiquitin and anti-DUSP6 antibodies (D). β-Actin was used as the loading control. ( E to G ) Overexpression of DUSP6 suppressed oxidation-induced increases in caspase-3 cleavage (E), apoptosis (F), and mitochondrial fragmentation (G). HeLa cells transiently transfected with either an empty control vector (−) or a vector encoding Flag-DUSP6 (+) were untreated or treated with 0.5 mM H 2 O 2 for 1 hour at 24 hours after transfection. In (E), cell lysates were subjected to IB with the indicated antibodies. In (A) to (E), blots are representatives of at least three independent experiments. In (F), cells were fixed for TUNEL staining (red) to identify apoptotic cells and immunofluorescence (IF) labeling of Flag (green) to assess Flag-DUSP6 expression. 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used for counterstaining of nuclei. Scale bar, 100 μm. Representative confocal images are shown. Quantification of TUNEL-positive (TUNEL + ) cells is shown at the right. In (G), after the H 2 O 2 treatment, cells were incubated with MitoTracker Red (200 nM) for 50 min at 37°C before fixation, IF for Flag, and DAPI labeling. Representative confocal images are shown. Scale bar, 10 μm. Black and white insets are magnifications of the boxed areas of MitoTracker labeling. Quantification of cells with different forms of mitochondrial morphology is shown at the right. Quantification data in (F) and (G) represent means ± SEM from n = 3 independent experiments. * P

    Journal: Science Advances

    Article Title: DUSP6 SUMOylation protects cells from oxidative damage via direct regulation of Drp1 dephosphorylation

    doi: 10.1126/sciadv.aaz0361

    Figure Lengend Snippet: H 2 O 2 destabilizes DUSP6 via ubiquitin-mediated degradation, and overexpression of DUSP6 protects cells against oxidative stress. ( A and B ) H 2 O 2 treatment decreased the levels of endogenous DUSP6 proteins in a dose- and time-dependent manner. Lysates from HeLa cells treated with H 2 O 2 at different concentrations for 1 hour (A) and at 0.5 mM for different times (B) as indicated were analyzed by immunoblotting (IB) using an anti-DUSP6 antibody. β-Actin was used as the loading control. ( C and D ) H 2 O 2 treatment increased caspase-3 cleavage (C) and DUSP6 ubiquitination (D). Lysates from HeLa cells untreated or treated with 0.5 mM H 2 O 2 for 1 hour were subjected to IB with anti-cleaved caspase-3 and anti-DUSP6 antibodies (C) or they were subjected to immunoprecipitation (IP) with the DUSP6 antibody [or control immunoglobulin G (IgG)] before being used for IB with anti-Ubiquitin and anti-DUSP6 antibodies (D). β-Actin was used as the loading control. ( E to G ) Overexpression of DUSP6 suppressed oxidation-induced increases in caspase-3 cleavage (E), apoptosis (F), and mitochondrial fragmentation (G). HeLa cells transiently transfected with either an empty control vector (−) or a vector encoding Flag-DUSP6 (+) were untreated or treated with 0.5 mM H 2 O 2 for 1 hour at 24 hours after transfection. In (E), cell lysates were subjected to IB with the indicated antibodies. In (A) to (E), blots are representatives of at least three independent experiments. In (F), cells were fixed for TUNEL staining (red) to identify apoptotic cells and immunofluorescence (IF) labeling of Flag (green) to assess Flag-DUSP6 expression. 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used for counterstaining of nuclei. Scale bar, 100 μm. Representative confocal images are shown. Quantification of TUNEL-positive (TUNEL + ) cells is shown at the right. In (G), after the H 2 O 2 treatment, cells were incubated with MitoTracker Red (200 nM) for 50 min at 37°C before fixation, IF for Flag, and DAPI labeling. Representative confocal images are shown. Scale bar, 10 μm. Black and white insets are magnifications of the boxed areas of MitoTracker labeling. Quantification of cells with different forms of mitochondrial morphology is shown at the right. Quantification data in (F) and (G) represent means ± SEM from n = 3 independent experiments. * P

    Article Snippet: Antibodies and reagents Anti-cleaved caspase-3 (1:1000; 9661), anti-ERK1/2 (1:1000; 9102), anti–p-ERK1/2 (1:1000; 9106), anti-SAE1 (1:1000; 8688), anti–p–Drp1-S616 (1:1000; 3455), and anti–p–Drp1-S637 (1:1000; 4867) were from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Over Expression, Immunoprecipitation, Transfection, Plasmid Preparation, TUNEL Assay, Staining, Immunofluorescence, Labeling, Expressing, Incubation