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  • 99
    Cell Signaling Technology Inc cleaved caspase 3
    (A) β-Galactosidase activity assay; HCT116 cells were assayed for β-galactosidase activity after treatment with 20 μM cisplatin or TriplatinNC for 96 h. Shown is a representative of two independent experiments. (B) Confocal microscopy; HCT116 cells were treated with 20 μM TriplatinNC for 24 h. Left, top panel; DAPI stained DNA (blue). Right, top panel; cleaved <t>caspase-3</t> (yellow). Bottom panel; merged DAPI and cleaved caspase-3. Apoptotic cell (white star) containing condensed/fragmented DNA and active caspase-3. Cells with compacted DNA (white arrows) do not contain active caspase-3. (C) Summary; percentage of HCT116 cells undergoing the indicated cellular process after treatment with 20 μM cisplatin or TriplatinNC for 24 h. (D) Schematic; TriplatinNC localizes to the cytoplasm and nucleolus of interphase cells. Cells treated while in S-G 2 proceed through mitosis. During prophase, the DNA (blue) condenses and the nuclear membrane (red) disintegrates allowing cytoplasmic pools of TriplatinNC to interact with condensed DNA. Cells undergo cytokinesis; however, the DNA does not decondense or progress through G 1 . (a) Determined by immunofluorescence: β-tubulin, nucleophosmin/B23, and DAPI DNA staining (Figure 5 A,B). (b) Determined by β-galactosidase staining and light microscopy at 200× magnification (panel A). (c) Determined by immunofluorescence; cleaved caspase-3; and DAPI DNA staining. The percentage of cells undergoing apoptosis was determined as the number of cells positive for cleaved caspase-3 divided by the total number of cells. n > 500 cells each for two repeat experiments (Figure 5 B).
    Cleaved Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 30782 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc caspase 3
    <t>BAX/CASP3</t> immunoreactivity in male placenta of CTR female baboons, n = 3 ( A , G ); male placenta of NR female baboon, n = 3 ( B , H ); preabsorbed negative CTR ( C , I ); female placenta of CTR female baboon, n = 4 ( D , J ); female placenta of NR female baboon, n =
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    Cell Signaling Technology Inc α cleaved caspase 3
    SEMA3B induces <t>caspase-3-dependent</t> apoptosis and VEGF 165 antagonizes this effect. ( A ) Flow cytometry analysis of apoptosis induction in H1299 cells 72 h posttransfection. NSCLC H1299 cells were transfected with pcDNA3 (vector control) SEMA3B, VEGF 121 (V121), SEMA3B plus V121 (SV121), SEMAMUT1, SEMAMUT2, VEGF 165 (V165), and SEMA3B plus V165 (SV165), and FACS assay was performed to determine induction of apoptosis. The number of tumor cells undergoing apoptosis are indicated by sub G 0 peak. ( B ) Caspase-3 cleavage increase detected by Western blotting as a hallmark of apoptotic activation. Vectors used in transfection of H1299 cells are as follows: lane 1 (vector control), lane 2 (SEMA3B), lane 3 (SEMAMUT1), lane 4 (SEMAMUT2), lane 5 (V121), lane 6 (SEMA3B plus V121), lane 7 (V165), and lane 8 (SEMA3B + V165).
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    Cell Signaling Technology Inc cleaved caspase 3 cc3
    BRCA1 and BRCA2 mutations result in γ H2AX foci accumulation in response to cisplatin treatment. Section of mice treated with a single dose of cisplatin or vehicle were stained for ( A ) γ H2AX and ( B ) <t>CC3.</t> BRCA mutant (mt) models display a significant increase in γ H2AX foci that was not observed in the WT models 24 h after treatment. Cleaved <t>caspase-3</t> staining, however, was not increased in the BRCA mt models. Error bars represent s.d.
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    Cell Signaling Technology Inc cleaved caspase 3 ccasp3
    BRCA1 and BRCA2 mutations result in γ H2AX foci accumulation in response to cisplatin treatment. Section of mice treated with a single dose of cisplatin or vehicle were stained for ( A ) γ H2AX and ( B ) <t>CC3.</t> BRCA mutant (mt) models display a significant increase in γ H2AX foci that was not observed in the WT models 24 h after treatment. Cleaved <t>caspase-3</t> staining, however, was not increased in the BRCA mt models. Error bars represent s.d.
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    Cell Signaling Technology Inc polyclonal cleaved caspase 3
    BRCA1 and BRCA2 mutations result in γ H2AX foci accumulation in response to cisplatin treatment. Section of mice treated with a single dose of cisplatin or vehicle were stained for ( A ) γ H2AX and ( B ) <t>CC3.</t> BRCA mutant (mt) models display a significant increase in γ H2AX foci that was not observed in the WT models 24 h after treatment. Cleaved <t>caspase-3</t> staining, however, was not increased in the BRCA mt models. Error bars represent s.d.
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    Cell Signaling Technology Inc phosphorylated cleaved caspase 3
    Assessment of cellular apoptosis. (A) Apoptotic hepatocytes in rat liver were identified by TUNEL assay. TUNEL (+) cells were quantified from 10 randomly selected fields at × 100. (B) Total and cleaved <t>caspase-3</t> proteins measured by western blotting.
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    Cell Signaling Technology Inc active cleaved caspase 3
    Metformin worsens hindlimb atrophy and hyperactive behavior and induces <t>caspase</t> 3 activation and tau cleavage. a - c Open field test in 5-month old P301S transgenic mice and age-matched WT non transgenic mice. Distance travelled ( a ), speed ( b ) and number of rearings ( c ) are shown. Bars represent the average values ± SEM. * p
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    Cell Signaling Technology Inc cleaved caspase 3 af488
    Metformin worsens hindlimb atrophy and hyperactive behavior and induces <t>caspase</t> 3 activation and tau cleavage. a - c Open field test in 5-month old P301S transgenic mice and age-matched WT non transgenic mice. Distance travelled ( a ), speed ( b ) and number of rearings ( c ) are shown. Bars represent the average values ± SEM. * p
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    Cell Signaling Technology Inc cleaved caspase 3 5a1e
    Metformin worsens hindlimb atrophy and hyperactive behavior and induces <t>caspase</t> 3 activation and tau cleavage. a - c Open field test in 5-month old P301S transgenic mice and age-matched WT non transgenic mice. Distance travelled ( a ), speed ( b ) and number of rearings ( c ) are shown. Bars represent the average values ± SEM. * p
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    Cell Signaling Technology Inc cleaved caspase 3 d3e9
    Metformin worsens hindlimb atrophy and hyperactive behavior and induces <t>caspase</t> 3 activation and tau cleavage. a - c Open field test in 5-month old P301S transgenic mice and age-matched WT non transgenic mice. Distance travelled ( a ), speed ( b ) and number of rearings ( c ) are shown. Bars represent the average values ± SEM. * p
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    Cell Signaling Technology Inc peptide
    Metformin worsens hindlimb atrophy and hyperactive behavior and induces <t>caspase</t> 3 activation and tau cleavage. a - c Open field test in 5-month old P301S transgenic mice and age-matched WT non transgenic mice. Distance travelled ( a ), speed ( b ) and number of rearings ( c ) are shown. Bars represent the average values ± SEM. * p
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    Cell Signaling Technology Inc cleaved caspase 3 tyr705
    Metformin worsens hindlimb atrophy and hyperactive behavior and induces <t>caspase</t> 3 activation and tau cleavage. a - c Open field test in 5-month old P301S transgenic mice and age-matched WT non transgenic mice. Distance travelled ( a ), speed ( b ) and number of rearings ( c ) are shown. Bars represent the average values ± SEM. * p
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    Cell Signaling Technology Inc cyclind1and cleaved caspase 3
    Metformin worsens hindlimb atrophy and hyperactive behavior and induces <t>caspase</t> 3 activation and tau cleavage. a - c Open field test in 5-month old P301S transgenic mice and age-matched WT non transgenic mice. Distance travelled ( a ), speed ( b ) and number of rearings ( c ) are shown. Bars represent the average values ± SEM. * p
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    Cell Signaling Technology Inc dual meca32 cleaved caspase 3
    Metformin worsens hindlimb atrophy and hyperactive behavior and induces <t>caspase</t> 3 activation and tau cleavage. a - c Open field test in 5-month old P301S transgenic mice and age-matched WT non transgenic mice. Distance travelled ( a ), speed ( b ) and number of rearings ( c ) are shown. Bars represent the average values ± SEM. * p
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    Cell Signaling Technology Inc human cleaved caspase 3
    Apoptotic cells visualized by staining for activated caspase 3 and caspase-cleaved cytokeratin Both nuclei and nuclear fragments are stained for activated caspase 3 in larger numbers in cardiac allgrafts to FcγRIII KO recipients (A) compared to WT recipients (B). Arterial and capillary deposits of C4d were more intense in cardiac allgrafts to FcγRIII KO recipients (C) compared to WT recipients (D).
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    Cell Signaling Technology Inc cleaved caspase 3 cl csp3
    Jasplakinolide (Jasplakinolide) attenuates isoflurane-mediated reduction in dendritic filopodial spines and enhancement of apoptosis in DIV4-7 neurons. Primary neurons (4-7 days in vitro – DIV4-7) were exposed to 1.4% isoflurane for 4 h with and without pre-treatment with the actin cytoskeleton stabilizer Jasplakinolide (1 h, 1 μM) and incubated with antibodies for drebrin (neuronal Factin binding protein) (A-C, quantitation shown in panel D), the apoptotic marker cleaved <t>caspase</t> 3 <t>(cl-Csp3)</t> (E-G, quantitation shown in panel 3H), and the nuclear marker DAPI. Pretreatment with Jasplakinolide significantly (C, n = 4-6; * p = 0.0144 vs . isoflurane) attenuated isoflurane-mediated decreased (B, # p = 0.0337 vs . basal) in drebrin immunofluorescence and significantly (n = 4-6; * p = 0.001 vs . isoflurane) decreased cl-Csp3 expression (G). Drebrin (green pixels) along dendrites is normalized to DAPI (blue pixels) and cl-Csp3 (red pixels) is normalized to DAPI. Scale bar, 10 μm. Error bars, standard error of the mean (s.e.m.).
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    Cell Signaling Technology Inc cleaved caspase 3 protein
    Characterization of 32 P uptake by the cell. A. 32 P is directly incorporated into cellular DNA. Mouse CRL2836 or human HeLa S3 cell lines were incubated overnight with 32 P[PO 4 ] and then grown for 48 h in non-radioactive medium (lanes 1 through 4), or grown for 24 h in non-radioactive medium, grown for 24 h with 32 P[PO 4 ], and then grown for 24 h in non-radioactive medium (lanes 5 through 8), or grown for 48 h in non-radioactive medium, then grown for 24 h with 32 P[PO 4 ] (lanes 9 through 12). The extracted nucleic acids were incubated with DNase I, the digestion products were run on a 5% polyacrylamide gel and exposed to film. B. Apoptosis induced by 32 P in mouse CRL2836 cells. Mouse CRL2836 cells were incubated with 0, 2.5, 5, 10 or 20 μCi 32 P[PO 4 ] for 24 h, and non-radioactive medium added for an additional 24 h. Protein was extracted from each well and analyzed for apoptosis by western blots using antibody to cleaved <t>caspase-3</t> protein (Lanes 1 through 5). Antibody against beta-actin was used to verify identical amounts of protein were loaded (lanes 6 through 10).
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    Cell Signaling Technology Inc cleaved caspase 3 staining
    Combination of oncolytic herpes simplex virus (oHSV) and S-TRAIL leads to <t>caspase-3/7-mediated</t> apoptosis in resistant glioblastoma multiforme (GBM) cells . ( a – d ) Caspase-3/7 activity and cell viability of ( a , c ) LN229 and ( b , d ) GBM8F treated with
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    Cell Signaling Technology Inc hunu cleaved caspase 3
    Transplanted ferumoxytol‐labeled human neural progenitor cells differentiate in the porcine spinal cord. Representative fluorescent micrographs of immunohistochemical staining with a mouse monoclonal anti‐human glial fibrillary acidic protein (GFAP) antibody (green) and a rabbit polyclonal anti‐human nestin antibody (red) in unlabeled human neural progenitor cells (hNPC) (A) , ferumoxytol‐labeled hNPC‐(F) Low (B) , and hNPC‐F High (C) grafts from postoperative day 105 are shown with high‐magnification insets. All nuclei are observed with 49,6‐diamidino‐2‐phenylindole staining (blue). Five grafts from each condition with > 10% engraftment were chosen for analysis. GFAP+ or nestin+ signal was not observed on the contralateral side or in rejected grafts. High‐magnification images of unlabeled hNPC ( D ), hNPC‐F Low ( E ), and hNPC‐F High ( F ) cell grafts expressing human nucleus <t>(HuNu+)</t> (green) and Ki67+ (red) show few proliferating transplanted cells (solid arrows). High‐magnification images of unlabeled hNPC (G) , hNPC‐F Low (H) , and hNPC‐F High (I) cell grafts expressing HuNu+ (green) and cleaved <t>caspase‐3</t> (CC3) (red) show few apoptotic transplanted cells (open arrows). The relative expression of GFAP+ and nestin+ was calculated for all groups (J‐L) . The relative expression of HuNu+ and Ki67+ was calculated (M) , as was that between HuNu+ and CC3 (N) . Scale bars = ×4, 500 μm (A‐C) ; ×100, 10 μm (A‐C) (insets); ×40, 25 μm (D‐I) . Graphs are displayed as mean ± SD. Abbreviations: CC3, cleaved caspase 3; DAPI, 4′,6‐diamidino‐2‐phenylindole; F, ferumoxytol; GFAP, glial fibrillary acidic protein; hNPC, human neural progenitor cell; HuNu, human nucleus; ns, not significant.
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    Cell Signaling Technology Inc myocardial cleaved caspase 3
    Effects of delayed isoflurane preconditioning on the expression of eNOS (A), iNOS(B), COX-2 (C), Cleaved <t>Caspase-3</t> (D) in rat hearts exposed to ischemia-reperfusion (IR). IR: ischemia reperfusion; Iso: isoflurane; TAC: Transverse aortic constriction. Data are mean ± SD, n = 6 hearts/group. *P
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    Cell Signaling Technology Inc rabbit cleaved caspase 3
    ) (A) Immunoblotting of spinal cord lysates from 4 SMAΔ7 mice and 3 wild-type mice at PND12. Spinal cords from SMAΔ7 mice show low expression of the full-length SMN protein, and higher levels of UPR markers cleaved Atf6 and Chop. (B) GUA treatment results in reduction of Ire1 , Chop , Atf4 and Pfdn5 mRNA expression in spinal cords of SMAΔ7 mice, to levels similar in control wild-type mice. (C) Decreased <t>Caspase-3</t> expression, as well as ER stress-associated Caspase-4 and Caspase-12 expression was observed in spinal cord lysates of SMAΔ7 mice treated with GUA. (D) While mRNA levels of Smn remain low in the SMAΔ7 animals, expression of the MN marker ChAT increases in the GUA-treated animals. (E) Western blot analysis of spinal cord lysates of SMAΔ7 and wild-type mice, treated with either DMSO or GUA, showing that GUA reduced UPR marker expression but led to increased MN markers Smi-32 and ChAT. (F) Representative images of ventral horn ChAT + MNs (red) of the respectively treated animals. Scale bar represents 100 μm. (G) Quantification of the number of ChAT + MNs in the ventral horns of each 10 μm section. 4–6 animals from each condition, and 20 lumbar sections representing the entire lumbar spinal cord were analyzed. Error bars are mean ± S.E.M. (H) Measurement of soma sizes of ChAT + ventral horn MNs indicating that the average MN size of SMAΔ7 animals treated with GUA is significantly larger than DMSO-treated SMAΔ7 animals. (I) Kaplan-Meier survival curve of SMAΔ7 mice treated with either DMSO (n = 9) or GUA (n = 8). Survival increased by approximately 35% (4 days) upon GUA treatment ( p = 0.0122). (J) Righting reflex time of DMSO and GUA-treated mutant and wild-type animals indicating that GUA-treated SMAΔ7 mice have significantly reduced righting reflex compared to DMSO controls ( p = 0.033). (K) Neuromuscular junctions (NMJs) of hindlimb muscles stained with α-bungarotoxin (BTX) and neurofilaments are identified by Smi-32 staining. Denervated NMJs are evident in DMSO-treated SMAΔ7 mice whereas the same region in GUA-treated SMAΔ7 animals showed preservation of NMJ innervation, similar to a wild-type animal. Magnified images of the white boxes are shown in the bottom panel. Scale bars represent 100 μm. (L) Analysis of innervation status of hindlimb NMJs in respective treated mice. NMJs are scored as innervated when there is Smi-32 staining of neurofilaments at the NMJs. GUA treatment in SMA mice led to a 20% increase in innervated NMJs ( p = 0.0125).
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    Cell Signaling Technology Inc cleaved caspase 3 elisa
    (a, b) Western blotting for the ratio of phosphorylated insulin receptor on tyrosine 1150/1151 (p-IR) and IRS-1 Ser307 (p-IRS-1) to total protein. (c) Western blotting for the ratio of Akt2 to β -actin. (d) <t>ELISA</t> results for cleaved <t>caspase</t> 3 levels. ∗ P
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    Cell Signaling Technology Inc cleaved caspase 3 antidody
    (a, b) Western blotting for the ratio of phosphorylated insulin receptor on tyrosine 1150/1151 (p-IR) and IRS-1 Ser307 (p-IRS-1) to total protein. (c) Western blotting for the ratio of Akt2 to β -actin. (d) <t>ELISA</t> results for cleaved <t>caspase</t> 3 levels. ∗ P
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    Cell Signaling Technology Inc cleaved caspase 3 antigen
    Therapeutic RDV but not LPV/RTV-IFNb diminishes signs of ALI. a Representative images of the histological features of acute lung injury 6 dpi comparing a mock-infected mouse to the therapeutic treatment groups described in Figs. 5 and 6 . Symbols identifying example features of disease are indicated in the figure. b American Thoracic Society Lung Injury Score derived as described in Fig. 3 . The numbers of animals per group quantitated: vehicle RDV N = 7, RDV N = 7, vehicle LPV/RTV-IFNb N = 9, LPV/RTV-IFNb low N = 7, LPV/RTV-IFNb high N = 8. c Diffuse alveolar damage score quantitating the degree of cellular sloughing, necrosis, and breakdown of barrier epithelium and vascular leakage. For both b and c , scores were blindly assessed in three random high power (×60) fields of diseased lung tissue sections. d Quantitation of cleaved <t>caspase-3</t> antigen staining in lung tissue sections from studies described in Figs. 5 –7. Cleaved caspase-3 is a marker of cell death. The numbers of animals per group quantitated for all groups was N = 5/group. For the box and whisker plots, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range. For b – d , asterisks indicate statistical significance by one-way ANOVA and Kruskal–Wallis multiple comparison test.
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    Cell Signaling Technology Inc cleaved caspase 3 c casp3
    Substitution of Matrigel/rCOL by hCOL from fibroblasts leads to lumenized EC network formation with involvement of apoptosis. ( a , b ) 3D hydrogel constructs containing GFP positive HUVECs and hASCs and hCOL after 14 days of cultivation in SFM. ( c ) Virtual stacks of GFP-HUVECs co-cultured with hASCs in hCOL for 14 days and stained for α-SMA. Nuclei were counterstained with DAPI. α-SMA positive cells are in physical contact with GFP-HUVECs. ( d ) Virtual stacks of HUVECs co-cultured with hASCs and incubated with Texas red-labeled Dextran. ( e – e ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM and incubated with Texas red-labeled dextran. Cryo-sections were stained with DAPI. ( e ) GFP-HUVEC, ( e ’) DAPI, ( e ”) Texas red-labeled dextran, ( e ”’) Superimposed image of ( e , e ’, e ”). Intense DAPI staining of fragmented DNA is localized within the lumen filled with Texas red-labeled dextran. ( f – f ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM. Cryo-sections were stained for cleaved caspase 3 and DAPI. ( f ) GFP-HUVEC, ( f ’) DAPI, ( f ”) cleaved caspase 3 <t>(CASP3),</t> ( f ”’) superimposed image of ( f , f ’, f ”). Intense DAPI staining of fragmented DNA is localized within the lumen and co-localizes with the signal for cleaved caspase 3. Scale bar: ( a ) 500 µm; ( b ) 200 µm; ( c – f ”’) 30 µm.
    Cleaved Caspase 3 C Casp3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 81/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Substitution of Matrigel/rCOL by hCOL from fibroblasts leads to lumenized EC network formation with involvement of apoptosis. ( a , b ) 3D hydrogel constructs containing GFP positive HUVECs and hASCs and hCOL after 14 days of cultivation in SFM. ( c ) Virtual stacks of GFP-HUVECs co-cultured with hASCs in hCOL for 14 days and stained for α-SMA. Nuclei were counterstained with DAPI. α-SMA positive cells are in physical contact with GFP-HUVECs. ( d ) Virtual stacks of HUVECs co-cultured with hASCs and incubated with Texas red-labeled Dextran. ( e – e ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM and incubated with Texas red-labeled dextran. Cryo-sections were stained with DAPI. ( e ) GFP-HUVEC, ( e ’) DAPI, ( e ”) Texas red-labeled dextran, ( e ”’) Superimposed image of ( e , e ’, e ”). Intense DAPI staining of fragmented DNA is localized within the lumen filled with Texas red-labeled dextran. ( f – f ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM. Cryo-sections were stained for cleaved caspase 3 and DAPI. ( f ) GFP-HUVEC, ( f ’) DAPI, ( f ”) cleaved caspase 3 <t>(CASP3),</t> ( f ”’) superimposed image of ( f , f ’, f ”). Intense DAPI staining of fragmented DNA is localized within the lumen and co-localizes with the signal for cleaved caspase 3. Scale bar: ( a ) 500 µm; ( b ) 200 µm; ( c – f ”’) 30 µm.
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    Cell Signaling Technology Inc af488 conjugated cleaved caspase 3
    Substitution of Matrigel/rCOL by hCOL from fibroblasts leads to lumenized EC network formation with involvement of apoptosis. ( a , b ) 3D hydrogel constructs containing GFP positive HUVECs and hASCs and hCOL after 14 days of cultivation in SFM. ( c ) Virtual stacks of GFP-HUVECs co-cultured with hASCs in hCOL for 14 days and stained for α-SMA. Nuclei were counterstained with DAPI. α-SMA positive cells are in physical contact with GFP-HUVECs. ( d ) Virtual stacks of HUVECs co-cultured with hASCs and incubated with Texas red-labeled Dextran. ( e – e ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM and incubated with Texas red-labeled dextran. Cryo-sections were stained with DAPI. ( e ) GFP-HUVEC, ( e ’) DAPI, ( e ”) Texas red-labeled dextran, ( e ”’) Superimposed image of ( e , e ’, e ”). Intense DAPI staining of fragmented DNA is localized within the lumen filled with Texas red-labeled dextran. ( f – f ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM. Cryo-sections were stained for cleaved caspase 3 and DAPI. ( f ) GFP-HUVEC, ( f ’) DAPI, ( f ”) cleaved caspase 3 <t>(CASP3),</t> ( f ”’) superimposed image of ( f , f ’, f ”). Intense DAPI staining of fragmented DNA is localized within the lumen and co-localizes with the signal for cleaved caspase 3. Scale bar: ( a ) 500 µm; ( b ) 200 µm; ( c – f ”’) 30 µm.
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    Cell Signaling Technology Inc apoptosis marker cleaved caspase 3
    Substitution of Matrigel/rCOL by hCOL from fibroblasts leads to lumenized EC network formation with involvement of apoptosis. ( a , b ) 3D hydrogel constructs containing GFP positive HUVECs and hASCs and hCOL after 14 days of cultivation in SFM. ( c ) Virtual stacks of GFP-HUVECs co-cultured with hASCs in hCOL for 14 days and stained for α-SMA. Nuclei were counterstained with DAPI. α-SMA positive cells are in physical contact with GFP-HUVECs. ( d ) Virtual stacks of HUVECs co-cultured with hASCs and incubated with Texas red-labeled Dextran. ( e – e ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM and incubated with Texas red-labeled dextran. Cryo-sections were stained with DAPI. ( e ) GFP-HUVEC, ( e ’) DAPI, ( e ”) Texas red-labeled dextran, ( e ”’) Superimposed image of ( e , e ’, e ”). Intense DAPI staining of fragmented DNA is localized within the lumen filled with Texas red-labeled dextran. ( f – f ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM. Cryo-sections were stained for cleaved caspase 3 and DAPI. ( f ) GFP-HUVEC, ( f ’) DAPI, ( f ”) cleaved caspase 3 <t>(CASP3),</t> ( f ”’) superimposed image of ( f , f ’, f ”). Intense DAPI staining of fragmented DNA is localized within the lumen and co-localizes with the signal for cleaved caspase 3. Scale bar: ( a ) 500 µm; ( b ) 200 µm; ( c – f ”’) 30 µm.
    Apoptosis Marker Cleaved Caspase 3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc cleaved caspase 3 cc3 antibody
    Substitution of Matrigel/rCOL by hCOL from fibroblasts leads to lumenized EC network formation with involvement of apoptosis. ( a , b ) 3D hydrogel constructs containing GFP positive HUVECs and hASCs and hCOL after 14 days of cultivation in SFM. ( c ) Virtual stacks of GFP-HUVECs co-cultured with hASCs in hCOL for 14 days and stained for α-SMA. Nuclei were counterstained with DAPI. α-SMA positive cells are in physical contact with GFP-HUVECs. ( d ) Virtual stacks of HUVECs co-cultured with hASCs and incubated with Texas red-labeled Dextran. ( e – e ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM and incubated with Texas red-labeled dextran. Cryo-sections were stained with DAPI. ( e ) GFP-HUVEC, ( e ’) DAPI, ( e ”) Texas red-labeled dextran, ( e ”’) Superimposed image of ( e , e ’, e ”). Intense DAPI staining of fragmented DNA is localized within the lumen filled with Texas red-labeled dextran. ( f – f ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM. Cryo-sections were stained for cleaved caspase 3 and DAPI. ( f ) GFP-HUVEC, ( f ’) DAPI, ( f ”) cleaved caspase 3 <t>(CASP3),</t> ( f ”’) superimposed image of ( f , f ’, f ”). Intense DAPI staining of fragmented DNA is localized within the lumen and co-localizes with the signal for cleaved caspase 3. Scale bar: ( a ) 500 µm; ( b ) 200 µm; ( c – f ”’) 30 µm.
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    Substitution of Matrigel/rCOL by hCOL from fibroblasts leads to lumenized EC network formation with involvement of apoptosis. ( a , b ) 3D hydrogel constructs containing GFP positive HUVECs and hASCs and hCOL after 14 days of cultivation in SFM. ( c ) Virtual stacks of GFP-HUVECs co-cultured with hASCs in hCOL for 14 days and stained for α-SMA. Nuclei were counterstained with DAPI. α-SMA positive cells are in physical contact with GFP-HUVECs. ( d ) Virtual stacks of HUVECs co-cultured with hASCs and incubated with Texas red-labeled Dextran. ( e – e ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM and incubated with Texas red-labeled dextran. Cryo-sections were stained with DAPI. ( e ) GFP-HUVEC, ( e ’) DAPI, ( e ”) Texas red-labeled dextran, ( e ”’) Superimposed image of ( e , e ’, e ”). Intense DAPI staining of fragmented DNA is localized within the lumen filled with Texas red-labeled dextran. ( f – f ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM. Cryo-sections were stained for cleaved caspase 3 and DAPI. ( f ) GFP-HUVEC, ( f ’) DAPI, ( f ”) cleaved caspase 3 <t>(CASP3),</t> ( f ”’) superimposed image of ( f , f ’, f ”). Intense DAPI staining of fragmented DNA is localized within the lumen and co-localizes with the signal for cleaved caspase 3. Scale bar: ( a ) 500 µm; ( b ) 200 µm; ( c – f ”’) 30 µm.
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    Substitution of Matrigel/rCOL by hCOL from fibroblasts leads to lumenized EC network formation with involvement of apoptosis. ( a , b ) 3D hydrogel constructs containing GFP positive HUVECs and hASCs and hCOL after 14 days of cultivation in SFM. ( c ) Virtual stacks of GFP-HUVECs co-cultured with hASCs in hCOL for 14 days and stained for α-SMA. Nuclei were counterstained with DAPI. α-SMA positive cells are in physical contact with GFP-HUVECs. ( d ) Virtual stacks of HUVECs co-cultured with hASCs and incubated with Texas red-labeled Dextran. ( e – e ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM and incubated with Texas red-labeled dextran. Cryo-sections were stained with DAPI. ( e ) GFP-HUVEC, ( e ’) DAPI, ( e ”) Texas red-labeled dextran, ( e ”’) Superimposed image of ( e , e ’, e ”). Intense DAPI staining of fragmented DNA is localized within the lumen filled with Texas red-labeled dextran. ( f – f ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM. Cryo-sections were stained for cleaved caspase 3 and DAPI. ( f ) GFP-HUVEC, ( f ’) DAPI, ( f ”) cleaved caspase 3 <t>(CASP3),</t> ( f ”’) superimposed image of ( f , f ’, f ”). Intense DAPI staining of fragmented DNA is localized within the lumen and co-localizes with the signal for cleaved caspase 3. Scale bar: ( a ) 500 µm; ( b ) 200 µm; ( c – f ”’) 30 µm.
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    Image Search Results


    (A) β-Galactosidase activity assay; HCT116 cells were assayed for β-galactosidase activity after treatment with 20 μM cisplatin or TriplatinNC for 96 h. Shown is a representative of two independent experiments. (B) Confocal microscopy; HCT116 cells were treated with 20 μM TriplatinNC for 24 h. Left, top panel; DAPI stained DNA (blue). Right, top panel; cleaved caspase-3 (yellow). Bottom panel; merged DAPI and cleaved caspase-3. Apoptotic cell (white star) containing condensed/fragmented DNA and active caspase-3. Cells with compacted DNA (white arrows) do not contain active caspase-3. (C) Summary; percentage of HCT116 cells undergoing the indicated cellular process after treatment with 20 μM cisplatin or TriplatinNC for 24 h. (D) Schematic; TriplatinNC localizes to the cytoplasm and nucleolus of interphase cells. Cells treated while in S-G 2 proceed through mitosis. During prophase, the DNA (blue) condenses and the nuclear membrane (red) disintegrates allowing cytoplasmic pools of TriplatinNC to interact with condensed DNA. Cells undergo cytokinesis; however, the DNA does not decondense or progress through G 1 . (a) Determined by immunofluorescence: β-tubulin, nucleophosmin/B23, and DAPI DNA staining (Figure 5 A,B). (b) Determined by β-galactosidase staining and light microscopy at 200× magnification (panel A). (c) Determined by immunofluorescence; cleaved caspase-3; and DAPI DNA staining. The percentage of cells undergoing apoptosis was determined as the number of cells positive for cleaved caspase-3 divided by the total number of cells. n > 500 cells each for two repeat experiments (Figure 5 B).

    Journal: Molecular Pharmaceutics

    Article Title: Nucleolar Targeting by Platinum: p53-Independent Apoptosis Follows rRNA Inhibition, Cell-Cycle Arrest, and DNA Compaction

    doi: 10.1021/mp5006867

    Figure Lengend Snippet: (A) β-Galactosidase activity assay; HCT116 cells were assayed for β-galactosidase activity after treatment with 20 μM cisplatin or TriplatinNC for 96 h. Shown is a representative of two independent experiments. (B) Confocal microscopy; HCT116 cells were treated with 20 μM TriplatinNC for 24 h. Left, top panel; DAPI stained DNA (blue). Right, top panel; cleaved caspase-3 (yellow). Bottom panel; merged DAPI and cleaved caspase-3. Apoptotic cell (white star) containing condensed/fragmented DNA and active caspase-3. Cells with compacted DNA (white arrows) do not contain active caspase-3. (C) Summary; percentage of HCT116 cells undergoing the indicated cellular process after treatment with 20 μM cisplatin or TriplatinNC for 24 h. (D) Schematic; TriplatinNC localizes to the cytoplasm and nucleolus of interphase cells. Cells treated while in S-G 2 proceed through mitosis. During prophase, the DNA (blue) condenses and the nuclear membrane (red) disintegrates allowing cytoplasmic pools of TriplatinNC to interact with condensed DNA. Cells undergo cytokinesis; however, the DNA does not decondense or progress through G 1 . (a) Determined by immunofluorescence: β-tubulin, nucleophosmin/B23, and DAPI DNA staining (Figure 5 A,B). (b) Determined by β-galactosidase staining and light microscopy at 200× magnification (panel A). (c) Determined by immunofluorescence; cleaved caspase-3; and DAPI DNA staining. The percentage of cells undergoing apoptosis was determined as the number of cells positive for cleaved caspase-3 divided by the total number of cells. n > 500 cells each for two repeat experiments (Figure 5 B).

    Article Snippet: Then 1:50 dilution NPM/B23 (Santa Cruz, #sc-5564), 1:100 dilution alpha-Tubulin (Cell Signaling, #3873), or 1:100 dilution of cleaved caspase-3 (Cell signaling, # 9662) was added overnight at 4 °C.

    Techniques: Activity Assay, Confocal Microscopy, Staining, Immunofluorescence, Light Microscopy

    miR-181a sensitizes neuroblastoma cells to apoptosis induced by mitochondrial uncouplers A. Overexpression of Parkin restores the inhibitory effect of miR-181a on mitophagy. SH-SY5Y cells were transfected with miR-181a and Parkin plasmid as indicated. Cells were then cultured in the presence or absence of FCCP (10 μM) or CCCP (10 μM) for 12 h. Samples were collected for western blot to analyze the protein expression of TIM23, Parkin, MFN1 and Actin. B. SH-SY5Y cells transfected with miR-181a or NC were treated with 25 μM FCCP or CCCP for 24 h. Western blot was performed to analyze the status of cleaved caspase-3, cleaved PARP and Tubulin. C. Densitomeric analysis of cleaved caspase-3/Tubulin and cleaved PARP/Tubulin protein ratios. D. Flow cytometry analysis of sub G1 population in SH-SY5Y cells treated with FCCP or CCCP for 24 h. Data shown are means±s.d. from three independent experiments, *p

    Journal: Oncotarget

    Article Title: MicroRNA-181a suppresses parkin-mediated mitophagy and sensitizes neuroblastoma cells to mitochondrial uncoupler-induced apoptosis

    doi: 10.18632/oncotarget.9786

    Figure Lengend Snippet: miR-181a sensitizes neuroblastoma cells to apoptosis induced by mitochondrial uncouplers A. Overexpression of Parkin restores the inhibitory effect of miR-181a on mitophagy. SH-SY5Y cells were transfected with miR-181a and Parkin plasmid as indicated. Cells were then cultured in the presence or absence of FCCP (10 μM) or CCCP (10 μM) for 12 h. Samples were collected for western blot to analyze the protein expression of TIM23, Parkin, MFN1 and Actin. B. SH-SY5Y cells transfected with miR-181a or NC were treated with 25 μM FCCP or CCCP for 24 h. Western blot was performed to analyze the status of cleaved caspase-3, cleaved PARP and Tubulin. C. Densitomeric analysis of cleaved caspase-3/Tubulin and cleaved PARP/Tubulin protein ratios. D. Flow cytometry analysis of sub G1 population in SH-SY5Y cells treated with FCCP or CCCP for 24 h. Data shown are means±s.d. from three independent experiments, *p

    Article Snippet: Primary antibodies used for western blotting were as follows: Parkin (Cell Signaling, 4211), LC3B (Sigma, L7543), MFN1 (Abcam, ab57602), TIM23 (Proteintech, 11123-1-AP), COXIV (Cell Signaling, 4844), C-III core 1 (Invitrogen, 459140), p62 (MBL, PM045), cleaved Caspase-3 (Cell signaling Technology, 9664), cleaved PARP (Cell signaling Technology, 5625), Actin (Proteintech, 60008-1-IG), α-Tubulin (Sigma, T9026).

    Techniques: Over Expression, Transfection, Plasmid Preparation, Cell Culture, Western Blot, Expressing, Flow Cytometry, Cytometry

    α-LA reduces the release of cytochrome C and the expression of cleaved caspase-3. Osteoblasts (5 × 10 6 ) were preincubated with α-LA for 12 h before treatment with 50 μM antimycin A (AMA) for 24 h. Osteoblasts were preincubated with α-LA (100 μM) for 12 h before treatment with 50 μM antimycin A (AMA) for 24 h. a Representative images of western blot analysis and quantitative analysis for cytochrome C in the cytosolic fractions. COX-4 was used as a control for the fractionation efficiency. b Representative images of western blot analysis and quantitative analysis for cleaved caspase-3 (* P

    Journal: Cell Stress & Chaperones

    Article Title: Protective effect of α-lipoic acid against antimycin A cytotoxicity in MC3T3-E1 osteoblastic cells

    doi: 10.1007/s12192-016-0735-z

    Figure Lengend Snippet: α-LA reduces the release of cytochrome C and the expression of cleaved caspase-3. Osteoblasts (5 × 10 6 ) were preincubated with α-LA for 12 h before treatment with 50 μM antimycin A (AMA) for 24 h. Osteoblasts were preincubated with α-LA (100 μM) for 12 h before treatment with 50 μM antimycin A (AMA) for 24 h. a Representative images of western blot analysis and quantitative analysis for cytochrome C in the cytosolic fractions. COX-4 was used as a control for the fractionation efficiency. b Representative images of western blot analysis and quantitative analysis for cleaved caspase-3 (* P

    Article Snippet: The primary antibodies against cleaved caspase-3 (no. 9579), p-creb (no. 9198), p-Akt (no. 4060), and total Akt (no. 4691) were obtained from Cell Signaling Technology, USA.

    Techniques: Expressing, Western Blot, Fractionation

    Inhibition of both PLK1 and EGFR is more effective than inhibition of either target alone in ER NSCLC xenograft models. Mice bearing PC9-ER9 xenograft tumors with volumes of 150 mm 3  were treated with volasertib and/or erlotinib or with vehicle controls ( A ) Tumor volume was measured twice weekly and significantly decreased after combination treatment. After 3 weeks of treatment, the mice were humanely killed. Resected tumors were subjected to immunohistochemical analysis for Ki67 ( C ) and caspase 3 ( B ) protein expression, which was scored and quantitated.

    Journal: Oncotarget

    Article Title: Polo-like kinase 1 inhibition diminishes acquired resistance to epidermal growth factor receptor inhibition in non-small cell lung cancer with T790M mutations

    doi: 10.18632/oncotarget.10332

    Figure Lengend Snippet: Inhibition of both PLK1 and EGFR is more effective than inhibition of either target alone in ER NSCLC xenograft models. Mice bearing PC9-ER9 xenograft tumors with volumes of 150 mm 3 were treated with volasertib and/or erlotinib or with vehicle controls ( A ) Tumor volume was measured twice weekly and significantly decreased after combination treatment. After 3 weeks of treatment, the mice were humanely killed. Resected tumors were subjected to immunohistochemical analysis for Ki67 ( C ) and caspase 3 ( B ) protein expression, which was scored and quantitated.

    Article Snippet: Additional control slides with positive and negative cell pellets were employed for cleaved caspase-3 (Cell Signaling Technology, cat. #8104).

    Techniques: Inhibition, Mouse Assay, Immunohistochemistry, Expressing

    Effect of trehalose on apoptosis and ERS in cryopreserved valve cells. (A and B) Western blotting using a cleaved caspase-3 antibody on cryopreserved valve cells treated with trehalose (0.1 mol/L) or DMSO for 16 weeks. (C, D and E) Western blotting using GRP78 and CHOP antibodies on cryopreserved valves treated with trehalose (0.1 mol/L) for 16 weeks. Data are expressed as the mean ± SD ( n = 20 per group). ** P

    Journal: PLoS ONE

    Article Title: The cryoprotectant trehalose could inhibit ERS-induced apoptosis by activating autophagy in cryoprotected rat valves

    doi: 10.1371/journal.pone.0194078

    Figure Lengend Snippet: Effect of trehalose on apoptosis and ERS in cryopreserved valve cells. (A and B) Western blotting using a cleaved caspase-3 antibody on cryopreserved valve cells treated with trehalose (0.1 mol/L) or DMSO for 16 weeks. (C, D and E) Western blotting using GRP78 and CHOP antibodies on cryopreserved valves treated with trehalose (0.1 mol/L) for 16 weeks. Data are expressed as the mean ± SD ( n = 20 per group). ** P

    Article Snippet: The following primary antibodies were used in the assays: antibodies for cleaved caspase-3 (no. 9654; Cell Signaling Technology, Danvers, MA, USA), GRP78 (no. 3183; Cell Signaling Technology), CHOP (no. 2895; Cell Signaling Technology), LC3A/B II/I (no. 12741; Cell Signaling Technology), p62 (no. 5114; Cell Signaling Technology), ATG-5 (no. 12994; Cell Signaling Technology), ATG-7 (no. 8558; Cell Signaling Technology), mTOR (no. 2983; Cell Signaling Technology), p-mTOR (Ser2448) (no. 5536; Cell Signaling Technology), p38 MAPK (no. 9212; Cell Signaling Technology), and p-p38 MAPK (no. 4511; Cell Signaling Technology), and β-actin (no. 3700; Cell Signaling Technology) was chosen as loading control.

    Techniques: Western Blot

    Effects of oleic acid (OLA) on the levels of cleaved apoptotic proteins (caspase-3, PARP) in palmitic acid (PAM)-treated AR42J cells. Cells were incubated with 250 µM OLA for 6~48 h in the presence or absence of 250 µM PAM. The levels of cleaved caspase-3 (C-Casp3) and cleaved PARP (C-PARP) were detected by Western blotting. The results on the right are presented as means±SEM of 3 independent experiments. The expression level of each protein was normalized to that of a housekeeping protein, β-actin. * p

    Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

    Article Title: Protective Effects of Oleic Acid Against Palmitic Acid-Induced Apoptosis in Pancreatic AR42J Cells and Its Mechanisms

    doi: 10.4196/kjpp.2013.17.1.43

    Figure Lengend Snippet: Effects of oleic acid (OLA) on the levels of cleaved apoptotic proteins (caspase-3, PARP) in palmitic acid (PAM)-treated AR42J cells. Cells were incubated with 250 µM OLA for 6~48 h in the presence or absence of 250 µM PAM. The levels of cleaved caspase-3 (C-Casp3) and cleaved PARP (C-PARP) were detected by Western blotting. The results on the right are presented as means±SEM of 3 independent experiments. The expression level of each protein was normalized to that of a housekeeping protein, β-actin. * p

    Article Snippet: The membranes were incubated with 5% non-fat dry milk, and then incubated with antibodies towards Bcl-2, Bcl-xL, Mcl-1, Bak, cleaved caspase-3 (Asp175), and cleaved PARP (Asp214) for overnight at 4℃ Membranes were incubated with appropriate horseradish peroxidase-linked secondary antibody (Cell Signaling, Danvers, USA) at a dilution of 1:2,500.

    Techniques: Incubation, Western Blot, Expressing

    (A) Western blot analysis of cleaved caspase-3 in cell lines following treatment with 6 Gy of radiation (IR), olaparib (500 nM), and/or PARP-1 shRNA (sh.PARP-1). Actin blots serve as loading control. (B) Photos of immunofluorescence staining for TUNEL-positive

    Journal: Molecular cancer therapeutics

    Article Title: Combining poly(ADP-ribose) polymerase 1 (PARP-1) inhibition and radiation in Ewing sarcoma results in lethal DNA damage

    doi: 10.1158/1535-7163.MCT-13-0338

    Figure Lengend Snippet: (A) Western blot analysis of cleaved caspase-3 in cell lines following treatment with 6 Gy of radiation (IR), olaparib (500 nM), and/or PARP-1 shRNA (sh.PARP-1). Actin blots serve as loading control. (B) Photos of immunofluorescence staining for TUNEL-positive

    Article Snippet: Antibodies used were anti-γH2AX (Millipore), m cleaved caspase-3 (Cell Signaling), anti-TUNEL (ApoptoTag Peroxidase kit, Millipore), and anti-PCNA (Santa Cruz Biotechnology).

    Techniques: Western Blot, shRNA, Immunofluorescence, Staining, TUNEL Assay

    25-HC suppresses myocardial apoptosis in IR hearts. (A) Representative images of TUNEL staining showing cardiac cell apoptosis. (B) The percentage of apoptotic cells was shown. (C) The protein levels of T-caspase-3, Bax, Bcl-2, and PARP1 in left ventricular tissues. (D) Statistical results were shown. (E) The mRNA levels of TNF-α, IL-6, and IL-1β in left ventricular tissues. Mean ± SEM. N = 5-6 per group, *P

    Journal: International Journal of Biological Sciences

    Article Title: 25-Hydroxycholesterol protects against myocardial ischemia-reperfusion injury via inhibiting PARP activity

    doi: 10.7150/ijbs.35075

    Figure Lengend Snippet: 25-HC suppresses myocardial apoptosis in IR hearts. (A) Representative images of TUNEL staining showing cardiac cell apoptosis. (B) The percentage of apoptotic cells was shown. (C) The protein levels of T-caspase-3, Bax, Bcl-2, and PARP1 in left ventricular tissues. (D) Statistical results were shown. (E) The mRNA levels of TNF-α, IL-6, and IL-1β in left ventricular tissues. Mean ± SEM. N = 5-6 per group, *P

    Article Snippet: Then, they were transferred on polyvinylidene fluoride membranes (Millipore, USA), followed by overnight incubation with the following primary antibodies: P38 antibody (1:1000, Proteintech), JNK antibody (1:1000, Proteintech), ERK1/2 antibody (1:1000, Proteintech), phospho-p38 MAPK antibody (1:1000, CST), phospho-SAPK/JNK antibody (1:1000, CST), phospho-p44/p42 MAPK (Erk1/2) antibody (1:1000, CST), Bax antibody (1:1000, CST), BCL2 antibody (1:1000, CST), caspase-3 antibody (1:1000, Proteintech), cleaved caspase-3 antibody (1:1000, CST), poly(ADP-ribose) polymerase1 (parp1) antibody (1:1000, CST), GAPDH antibody (1:10000, Abcam), and anti-poly(ADP-ribose) polymer (PAR) antibody (1:1000, CST).

    Techniques: TUNEL Assay, Staining

    The treatment of DBT mitigates Aβ-triggered caspase 3/9 levels. Cultured cortical neurons were co-treated with a series amount of DBT (DBT-L: 12.5 μg/mL; DBT-H: 100 μg/mL) and Aβ (20 μM) for 48 h. Then cells were labeled with caspase 3/9 detection kit and Hoechst for 30 min. The activities of caspase 3/9 were evaluated by measuring the fluorescence intensity. Micrographs were taken by a confocal microscope

    Journal: BMC Complementary and Alternative Medicine

    Article Title: Danggui Buxue Tang, an ancient Chinese herbal decoction, protects β-amyloid-induced cell death in cultured cortical neurons

    doi: 10.1186/s12906-018-2411-6

    Figure Lengend Snippet: The treatment of DBT mitigates Aβ-triggered caspase 3/9 levels. Cultured cortical neurons were co-treated with a series amount of DBT (DBT-L: 12.5 μg/mL; DBT-H: 100 μg/mL) and Aβ (20 μM) for 48 h. Then cells were labeled with caspase 3/9 detection kit and Hoechst for 30 min. The activities of caspase 3/9 were evaluated by measuring the fluorescence intensity. Micrographs were taken by a confocal microscope

    Article Snippet: The membranes were incubated with anti-Bcl2, Bax, cleaved- PARP and cleaved-caspase 3/9 (CST, Danvers, MA) at 1: 1000 dilutions, respectively, and anti-β-actin (Abcam, Cambridge, MA) at 1: 10,000 dilutions were kept for over 12 h at cold room.

    Techniques: Cell Culture, Labeling, Fluorescence, Microscopy

    A K-RAS mouse model of lung cancer but not a PTEN -null model of prostate cancer is sensitive to DR a , Representative images of lungs and average diameter of tumour nodules on the surface of the lungs (graph) of 7-week-old K-RAS LA2 ; P53 LSL/ WT mice under AL or DR conditions (n=3). b-d , H E staining (b) and immunohistochemical analyses of Ki-67 (c) and cleaved caspase-3 (d) in sections prepared from lungs in a . e-h , H E staining (e) and immunohistochemical analyses of phospho-Akt S473 (f), Ki-67 (g), and cleaved caspase-3 (h) in prostates of 11-week-old Probasin-Cre; PTEN L/L mice under AL or DR conditions. In c, d, g, and h , graphs to right of respective images indicate percent of total cells that are positive for Ki-67 or cleaved caspase-3. Graphs show means ± s.e.m. of percent of proliferating (c and g) or apoptotic cells (d and h) measured in 10 images (1000 nuclei counted per image) from 2 different tumours per group. All pictures were captured under the same magnification and scale bar = 20 µm. ** indicates P = 8.3 × 10 −8 .

    Journal: Nature

    Article Title: Tumours with PI3K activation are resistant to dietary restriction

    doi: 10.1038/nature07782

    Figure Lengend Snippet: A K-RAS mouse model of lung cancer but not a PTEN -null model of prostate cancer is sensitive to DR a , Representative images of lungs and average diameter of tumour nodules on the surface of the lungs (graph) of 7-week-old K-RAS LA2 ; P53 LSL/ WT mice under AL or DR conditions (n=3). b-d , H E staining (b) and immunohistochemical analyses of Ki-67 (c) and cleaved caspase-3 (d) in sections prepared from lungs in a . e-h , H E staining (e) and immunohistochemical analyses of phospho-Akt S473 (f), Ki-67 (g), and cleaved caspase-3 (h) in prostates of 11-week-old Probasin-Cre; PTEN L/L mice under AL or DR conditions. In c, d, g, and h , graphs to right of respective images indicate percent of total cells that are positive for Ki-67 or cleaved caspase-3. Graphs show means ± s.e.m. of percent of proliferating (c and g) or apoptotic cells (d and h) measured in 10 images (1000 nuclei counted per image) from 2 different tumours per group. All pictures were captured under the same magnification and scale bar = 20 µm. ** indicates P = 8.3 × 10 −8 .

    Article Snippet: Immunohistochemistry detection of PTEN, FOXO1 and cleaved caspase-3 antibodies (1:100, Cell Signaling Technologies) was performed on paraffin-embedded, sliced tumour sections according to the manufacturer’s protocols.

    Techniques: Mouse Assay, Staining, Immunohistochemistry

    Effects of modulation of PI3K signaling on the apoptotic response of tumours to DR a-d , Immunohistochemical analyses of FOXO1 (a, b) and cleaved caspase-3 (c, d) in tumours formed by DLD-WT and DLD-Mut cells and by the PTEN-inducible U87-MG cells in mice treated or non-treated with doxycycline (Dox). Graphs to right of images indicate percent of total cells that are positive for cleaved caspase-3. Data in c and d graphs are means ± s.e.m, measured in 9 images (1000 nuclei counted per image) from 3 different tumours per group. ** indicates P ≤ 0.01. All images were acquired at the same magnification and scale bar = 20 µm. Framed inserts in a and b are a 3.9-fold magnification of a representative area of the corresponding larger image. Arrows point to immunoreactivity for FOXO1 (a, b) or cleaved caspase-3 (c and d).

    Journal: Nature

    Article Title: Tumours with PI3K activation are resistant to dietary restriction

    doi: 10.1038/nature07782

    Figure Lengend Snippet: Effects of modulation of PI3K signaling on the apoptotic response of tumours to DR a-d , Immunohistochemical analyses of FOXO1 (a, b) and cleaved caspase-3 (c, d) in tumours formed by DLD-WT and DLD-Mut cells and by the PTEN-inducible U87-MG cells in mice treated or non-treated with doxycycline (Dox). Graphs to right of images indicate percent of total cells that are positive for cleaved caspase-3. Data in c and d graphs are means ± s.e.m, measured in 9 images (1000 nuclei counted per image) from 3 different tumours per group. ** indicates P ≤ 0.01. All images were acquired at the same magnification and scale bar = 20 µm. Framed inserts in a and b are a 3.9-fold magnification of a representative area of the corresponding larger image. Arrows point to immunoreactivity for FOXO1 (a, b) or cleaved caspase-3 (c and d).

    Article Snippet: Immunohistochemistry detection of PTEN, FOXO1 and cleaved caspase-3 antibodies (1:100, Cell Signaling Technologies) was performed on paraffin-embedded, sliced tumour sections according to the manufacturer’s protocols.

    Techniques: Immunohistochemistry, Mouse Assay

    IKK2 deficiency differentially affects gene expression of naive and arthritic SFs. a Representative immunodetection of IKK2 levels in TAT-Cre treated Ikk2 f/f ( Ikk2 Δ/Δ ) and TAT-Cre treated hTNFtg Ikk2 f/f ( hTNFtg Ikk2 Δ/Δ ) SFs compared to non-treated control cultures Ikk2 f/f and hTNFtg Ikk2 f/f , respectively (upper panel). b Representative MTT incorporation assay in the indicated genotypes; hTNF quantitation in the supernatants of hTNFtg Ikk2 Δ/Δ (grey column) and control hTNFtg Ikk2 f/f SF cultures (black column) ( n = 7, 2 experiments); flow cytometric detection of Vcam-1 and Icam-1 levels in cultured hTNFtg Ikk2 f/f (black), hTNFtg Ikk2 Δ/Δ (grey) and control SFs ( Ikk2 f/f : dotted black and Ikk2 Δ/Δ : dotted grey) ( n = 4, 2 experiments). c Representative immunodetection of Casp8 (C8), Cleaved Casp3 (CC3), Parp1, Sod2, Xiap, cIAP-1 and cFLIP (L) in cell extracts of Ikk2 Δ/Δ , hTNFtg Ikk2 Δ/Δ and control SF cultures treated with TNF (20 ng/ml, 5 h) ( n = 10). d Relative gene expression of the NFκB-regulated genes Il6 , Mmp-3 , -13 , -14 , Timp1 , Il1b and Tnfsf11 (RankL) (normalized to b2m levels) in SFs of the indicated genotypes upon TNF (20 ng/ml, 5 h) ( n = 3–8). e Survival rates of Ikk2 Δ/Δ and hTNFtg Ikk2 Δ/Δ SFs treated with TNF, zVAD, Nec1s and GW806742X (GW) as indicated ( n = 3). f Representative immunodetection of pMlkl, Mlkl, Ripk3, cFLIP (S) and IKK2 levels in TNF-treated Ikk2 Δ/Δ , hTNFtg Ikk2 Δ/Δ and control SF cultures (TNF: 20 ng/ml, 5 h) ( n = 8, 4 experiments). Data are presented as the mean ± SEM. * P

    Journal: Nature Communications

    Article Title: The p55TNFR-IKK2-Ripk3 axis orchestrates arthritis by regulating death and inflammatory pathways in synovial fibroblasts

    doi: 10.1038/s41467-018-02935-4

    Figure Lengend Snippet: IKK2 deficiency differentially affects gene expression of naive and arthritic SFs. a Representative immunodetection of IKK2 levels in TAT-Cre treated Ikk2 f/f ( Ikk2 Δ/Δ ) and TAT-Cre treated hTNFtg Ikk2 f/f ( hTNFtg Ikk2 Δ/Δ ) SFs compared to non-treated control cultures Ikk2 f/f and hTNFtg Ikk2 f/f , respectively (upper panel). b Representative MTT incorporation assay in the indicated genotypes; hTNF quantitation in the supernatants of hTNFtg Ikk2 Δ/Δ (grey column) and control hTNFtg Ikk2 f/f SF cultures (black column) ( n = 7, 2 experiments); flow cytometric detection of Vcam-1 and Icam-1 levels in cultured hTNFtg Ikk2 f/f (black), hTNFtg Ikk2 Δ/Δ (grey) and control SFs ( Ikk2 f/f : dotted black and Ikk2 Δ/Δ : dotted grey) ( n = 4, 2 experiments). c Representative immunodetection of Casp8 (C8), Cleaved Casp3 (CC3), Parp1, Sod2, Xiap, cIAP-1 and cFLIP (L) in cell extracts of Ikk2 Δ/Δ , hTNFtg Ikk2 Δ/Δ and control SF cultures treated with TNF (20 ng/ml, 5 h) ( n = 10). d Relative gene expression of the NFκB-regulated genes Il6 , Mmp-3 , -13 , -14 , Timp1 , Il1b and Tnfsf11 (RankL) (normalized to b2m levels) in SFs of the indicated genotypes upon TNF (20 ng/ml, 5 h) ( n = 3–8). e Survival rates of Ikk2 Δ/Δ and hTNFtg Ikk2 Δ/Δ SFs treated with TNF, zVAD, Nec1s and GW806742X (GW) as indicated ( n = 3). f Representative immunodetection of pMlkl, Mlkl, Ripk3, cFLIP (S) and IKK2 levels in TNF-treated Ikk2 Δ/Δ , hTNFtg Ikk2 Δ/Δ and control SF cultures (TNF: 20 ng/ml, 5 h) ( n = 8, 4 experiments). Data are presented as the mean ± SEM. * P

    Article Snippet: Cleaved caspase-3 detection was performed according to Ab manufacturer's instructions [Cell Signalling; #9661; 1:200].

    Techniques: Expressing, Immunodetection, MTT Assay, Quantitation Assay, Flow Cytometry, Cell Culture

    Ripk3-mediated signals are indispensable for persisting synovitis in hTNFtg Ikk2 Ms-KO mice. a Representative histological images of H E-, TB- and TRAP-stained ankle joint sections of 8-week-old hTNFtg Ikk2 f/f Ripk3 −/− ( n = 11) and hTNFtg Ikk2 Ms-KO Ripk3 −/− mice ( n = 13) in comparison to hTNFtg Ikk2 Ms-KO and Ikk2 f/f mice. Note the differences in synovium and adjacent tissues in respective areas indicated by the arrows. b Survival rates of Ikk2 Δ/Δ Ripk3 −/− and hTNFtg Ikk2 Δ/Δ Ripk3 −/− SF cultures treated with TNF (T), zVAD (Z), and nec-1 (N) as indicated. c Representative immunodetection of pMlkl, Mlkl and Ripk3 levels in the cell extracts of Ikk2 Δ/Δ Ripk3 −/− and hTNFtg Ikk2 Δ/Δ Ripk3 −/− SFs treated as indicated compared to control Ikk2 Δ/Δ Ripk3 +/+ and hTNFtg Ikk2 Δ/Δ Ripk3 +/+ samples, respectively ( n = 4 per genotype, 2 experiments). d Representative immunodetection of Casp8 (C8), Cleaved Casp3 (CC3), Xiap, cFLIP (s) and IKK2 levels in cell extracts of Ikk2 Δ/Δ Ripk3 −/− and hTNFtg Ikk2 Δ/Δ Ripk3 −/− SFs treated as indicated compared to control Ikk2 f/f Ripk3 −/− and hTNFtg Ikk2 f/f Ripk3 −/− samples, respectively (the non-hTNFtg control cultures are indicated as WT) ( n = 4 per genotype, 2 experiments). Scale bar: 500 μm. Data are presented as the mean ± SEM. *** P

    Journal: Nature Communications

    Article Title: The p55TNFR-IKK2-Ripk3 axis orchestrates arthritis by regulating death and inflammatory pathways in synovial fibroblasts

    doi: 10.1038/s41467-018-02935-4

    Figure Lengend Snippet: Ripk3-mediated signals are indispensable for persisting synovitis in hTNFtg Ikk2 Ms-KO mice. a Representative histological images of H E-, TB- and TRAP-stained ankle joint sections of 8-week-old hTNFtg Ikk2 f/f Ripk3 −/− ( n = 11) and hTNFtg Ikk2 Ms-KO Ripk3 −/− mice ( n = 13) in comparison to hTNFtg Ikk2 Ms-KO and Ikk2 f/f mice. Note the differences in synovium and adjacent tissues in respective areas indicated by the arrows. b Survival rates of Ikk2 Δ/Δ Ripk3 −/− and hTNFtg Ikk2 Δ/Δ Ripk3 −/− SF cultures treated with TNF (T), zVAD (Z), and nec-1 (N) as indicated. c Representative immunodetection of pMlkl, Mlkl and Ripk3 levels in the cell extracts of Ikk2 Δ/Δ Ripk3 −/− and hTNFtg Ikk2 Δ/Δ Ripk3 −/− SFs treated as indicated compared to control Ikk2 Δ/Δ Ripk3 +/+ and hTNFtg Ikk2 Δ/Δ Ripk3 +/+ samples, respectively ( n = 4 per genotype, 2 experiments). d Representative immunodetection of Casp8 (C8), Cleaved Casp3 (CC3), Xiap, cFLIP (s) and IKK2 levels in cell extracts of Ikk2 Δ/Δ Ripk3 −/− and hTNFtg Ikk2 Δ/Δ Ripk3 −/− SFs treated as indicated compared to control Ikk2 f/f Ripk3 −/− and hTNFtg Ikk2 f/f Ripk3 −/− samples, respectively (the non-hTNFtg control cultures are indicated as WT) ( n = 4 per genotype, 2 experiments). Scale bar: 500 μm. Data are presented as the mean ± SEM. *** P

    Article Snippet: Cleaved caspase-3 detection was performed according to Ab manufacturer's instructions [Cell Signalling; #9661; 1:200].

    Techniques: Mass Spectrometry, Mouse Assay, Staining, Immunodetection

    TMP decreases the gene expression and protein activity of the Akt signaling pathway and increases caspase-3 activity. (A) The reverse transcription-quantitative polymerase chain reaction revealed that TMP significantly downregulated the gene expression of Akt1, Akt2 and Akt3 at 1,600 and 3,200 µM. (B) Western blot analysis indicated that TMP decreased the activity of the Akt signaling pathway and increased the activity of caspase-3. β-actin was the loading control. (C) Quantification of western blot analysis results which validated that 1,600 and 3,200 µM significantly decreased the relative expression of p-Akt to t-Akt and increased the relative expression of cleaved-casp3 to t-casp3. *P

    Journal: Oncology Letters

    Article Title: Tetramethylpyrazine regulates breast cancer cell viability, migration, invasion and apoptosis by affecting the activity of Akt and caspase-3

    doi: 10.3892/ol.2018.7851

    Figure Lengend Snippet: TMP decreases the gene expression and protein activity of the Akt signaling pathway and increases caspase-3 activity. (A) The reverse transcription-quantitative polymerase chain reaction revealed that TMP significantly downregulated the gene expression of Akt1, Akt2 and Akt3 at 1,600 and 3,200 µM. (B) Western blot analysis indicated that TMP decreased the activity of the Akt signaling pathway and increased the activity of caspase-3. β-actin was the loading control. (C) Quantification of western blot analysis results which validated that 1,600 and 3,200 µM significantly decreased the relative expression of p-Akt to t-Akt and increased the relative expression of cleaved-casp3 to t-casp3. *P

    Article Snippet: SDS-PAGE (10% gel) was used to separate total-protein kinase B (t-Akt; cat. no. 4685), phosphorylated-Akt (p-Akt; cat. no. 4060), total-caspase-3 (t-casp3; cat. no. 9662) and β-actin (dilution of all antibodies, 1:1,000; cat. no. 4970; Cell Signaling Technology, Inc., Danvers, MA, USA), and SDS-PAGE (15% gel) was used to separate cleaved-caspase-3 (cleaved-casp3; dilution 1:1,000; cat. no. 9662; Cell Signaling Technology, Inc.).

    Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot

    BAX/CASP3 immunoreactivity in male placenta of CTR female baboons, n = 3 ( A , G ); male placenta of NR female baboon, n = 3 ( B , H ); preabsorbed negative CTR ( C , I ); female placenta of CTR female baboon, n = 4 ( D , J ); female placenta of NR female baboon, n =

    Journal: The Journal of Nutrition

    Article Title: Expression of the Placental Transcriptome in Maternal Nutrient Reduction in Baboons Is Dependent on Fetal Sex 1Expression of the Placental Transcriptome in Maternal Nutrient Reduction in Baboons Is Dependent on Fetal Sex 1 2Expression of the Placental Transcriptome in Maternal Nutrient Reduction in Baboons Is Dependent on Fetal Sex 1 2 3

    doi: 10.3945/jn.112.172148

    Figure Lengend Snippet: BAX/CASP3 immunoreactivity in male placenta of CTR female baboons, n = 3 ( A , G ); male placenta of NR female baboon, n = 3 ( B , H ); preabsorbed negative CTR ( C , I ); female placenta of CTR female baboon, n = 4 ( D , J ); female placenta of NR female baboon, n =

    Article Snippet: The primary antibodies and blocking peptides were as follows: caspase 3 (CASP3; Cell Signaling Technology, cat no. 9661, dilution 1:50) and blocking peptide (for negative CTR) (Cell Signaling Technology. cat no.1050, dilution 1:10); BCL2-associated X protein (BAX; Cell Signaling Technology, cat no. 2772, dilution 1:150), and blocking peptide (Cell Signaling Technology, cat no. 2772, dilution 1:30).

    Techniques:

    SEMA3B induces caspase-3-dependent apoptosis and VEGF 165 antagonizes this effect. ( A ) Flow cytometry analysis of apoptosis induction in H1299 cells 72 h posttransfection. NSCLC H1299 cells were transfected with pcDNA3 (vector control) SEMA3B, VEGF 121 (V121), SEMA3B plus V121 (SV121), SEMAMUT1, SEMAMUT2, VEGF 165 (V165), and SEMA3B plus V165 (SV165), and FACS assay was performed to determine induction of apoptosis. The number of tumor cells undergoing apoptosis are indicated by sub G 0 peak. ( B ) Caspase-3 cleavage increase detected by Western blotting as a hallmark of apoptotic activation. Vectors used in transfection of H1299 cells are as follows: lane 1 (vector control), lane 2 (SEMA3B), lane 3 (SEMAMUT1), lane 4 (SEMAMUT2), lane 5 (V121), lane 6 (SEMA3B plus V121), lane 7 (V165), and lane 8 (SEMA3B + V165).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Semaphorin 3B (SEMA3B) induces apoptosis in lung and breast cancer, whereas VEGF165 antagonizes this effect

    doi: 10.1073/pnas.0403969101

    Figure Lengend Snippet: SEMA3B induces caspase-3-dependent apoptosis and VEGF 165 antagonizes this effect. ( A ) Flow cytometry analysis of apoptosis induction in H1299 cells 72 h posttransfection. NSCLC H1299 cells were transfected with pcDNA3 (vector control) SEMA3B, VEGF 121 (V121), SEMA3B plus V121 (SV121), SEMAMUT1, SEMAMUT2, VEGF 165 (V165), and SEMA3B plus V165 (SV165), and FACS assay was performed to determine induction of apoptosis. The number of tumor cells undergoing apoptosis are indicated by sub G 0 peak. ( B ) Caspase-3 cleavage increase detected by Western blotting as a hallmark of apoptotic activation. Vectors used in transfection of H1299 cells are as follows: lane 1 (vector control), lane 2 (SEMA3B), lane 3 (SEMAMUT1), lane 4 (SEMAMUT2), lane 5 (V121), lane 6 (SEMA3B plus V121), lane 7 (V165), and lane 8 (SEMA3B + V165).

    Article Snippet: Pro-caspase 3 and cleaved caspase-3 Ab were purchased from Cell Signaling Technology (Beverly, MA).

    Techniques: Flow Cytometry, Cytometry, Transfection, Plasmid Preparation, FACS, Western Blot, Activation Assay

    BRCA1 and BRCA2 mutations result in γ H2AX foci accumulation in response to cisplatin treatment. Section of mice treated with a single dose of cisplatin or vehicle were stained for ( A ) γ H2AX and ( B ) CC3. BRCA mutant (mt) models display a significant increase in γ H2AX foci that was not observed in the WT models 24 h after treatment. Cleaved caspase-3 staining, however, was not increased in the BRCA mt models. Error bars represent s.d.

    Journal: British Journal of Cancer

    Article Title: BRCA1 and BRCA2 mutations sensitize to chemotherapy in patient-derived pancreatic cancer xenografts

    doi: 10.1038/bjc.2015.220

    Figure Lengend Snippet: BRCA1 and BRCA2 mutations result in γ H2AX foci accumulation in response to cisplatin treatment. Section of mice treated with a single dose of cisplatin or vehicle were stained for ( A ) γ H2AX and ( B ) CC3. BRCA mutant (mt) models display a significant increase in γ H2AX foci that was not observed in the WT models 24 h after treatment. Cleaved caspase-3 staining, however, was not increased in the BRCA mt models. Error bars represent s.d.

    Article Snippet: After antigen retrieval and serum block, sections were incubated at room temperature with γ H2AX (EMD Millipore, Etobicoke, ON, Canada, clone JBW301, 1:1000) and cleaved caspase-3 (CC3) (Cell Signaling, Danvers, MA, USA, #9661, 1/200) cocktail overnight.

    Techniques: Mouse Assay, Staining, Mutagenesis

    Assessment of cellular apoptosis. (A) Apoptotic hepatocytes in rat liver were identified by TUNEL assay. TUNEL (+) cells were quantified from 10 randomly selected fields at × 100. (B) Total and cleaved caspase-3 proteins measured by western blotting.

    Journal: Alcoholism, clinical and experimental research

    Article Title: Moderate alcohol consumption aggravates high-fat diet induced steatohepatitis in rats

    doi: 10.1111/j.1530-0277.2009.01122.x

    Figure Lengend Snippet: Assessment of cellular apoptosis. (A) Apoptotic hepatocytes in rat liver were identified by TUNEL assay. TUNEL (+) cells were quantified from 10 randomly selected fields at × 100. (B) Total and cleaved caspase-3 proteins measured by western blotting.

    Article Snippet: After blocking membrane, immunoblotting was performed based on the manufacturer's instruction for each primary antibody against total and phosphorylated cleaved caspase-3 (Cell Signaling Technology, Inc. MA), CYP2E1 (Chemicon International, Inc. MA), Bax and Bcl-2 (Santa Cruz Biotechnology, Inc. CA).

    Techniques: TUNEL Assay, Western Blot

    Metformin worsens hindlimb atrophy and hyperactive behavior and induces caspase 3 activation and tau cleavage. a - c Open field test in 5-month old P301S transgenic mice and age-matched WT non transgenic mice. Distance travelled ( a ), speed ( b ) and number of rearings ( c ) are shown. Bars represent the average values ± SEM. * p

    Journal: Molecular Neurodegeneration

    Article Title: Metformin promotes tau aggregation and exacerbates abnormal behavior in a mouse model of tauopathy

    doi: 10.1186/s13024-016-0082-7

    Figure Lengend Snippet: Metformin worsens hindlimb atrophy and hyperactive behavior and induces caspase 3 activation and tau cleavage. a - c Open field test in 5-month old P301S transgenic mice and age-matched WT non transgenic mice. Distance travelled ( a ), speed ( b ) and number of rearings ( c ) are shown. Bars represent the average values ± SEM. * p

    Article Snippet: Antibodies and reagents The following primary antibodies were used: mouse monoclonal antibodies against GSK3β (Millipore), IRβ, S6 (Santa Cruz), βI-tubulin (Sigma), β-actin (Sigma), caspase cleaved tau (Millipore), c-PARP, AKT (pan), IRS1 (Cell Signaling), PSD95 (Cell Signaling for Western blot or Millipore for immunocytochemistry), Synapsin-I (Millipore), GFP (Roche) and Rheb (Santa Cruz Biotechnologies); rabbit polyclonal antisera against p[Ser1162/1165]IRβ, p[S9]GSK3β, AMPK, p[T172]AMPK, PP2A, p[S240/244]S6, mTOR, p[S2448]mTOR, p[S473]AKT, active cleaved caspase 3 (Cell Signaling), actin (Sigma) and p[S616]IRS1 (Invitrogen), Synaptophysin (Cell Signaling).

    Techniques: Activation Assay, Transgenic Assay, Mouse Assay

    Apoptotic cells visualized by staining for activated caspase 3 and caspase-cleaved cytokeratin Both nuclei and nuclear fragments are stained for activated caspase 3 in larger numbers in cardiac allgrafts to FcγRIII KO recipients (A) compared to WT recipients (B). Arterial and capillary deposits of C4d were more intense in cardiac allgrafts to FcγRIII KO recipients (C) compared to WT recipients (D).

    Journal: Transplantation

    Article Title: The absence of FcγRIII results in increased pro-inflammatory response in FcγRIII-KO cardiac recipients

    doi: 10.1097/TP.0b013e31829c2455

    Figure Lengend Snippet: Apoptotic cells visualized by staining for activated caspase 3 and caspase-cleaved cytokeratin Both nuclei and nuclear fragments are stained for activated caspase 3 in larger numbers in cardiac allgrafts to FcγRIII KO recipients (A) compared to WT recipients (B). Arterial and capillary deposits of C4d were more intense in cardiac allgrafts to FcγRIII KO recipients (C) compared to WT recipients (D).

    Article Snippet: Slides were incubated for 30 minutes in a serum free blocking solution (Dako, Carpenteria, CA) followed by an overnight incubation with a monoclonal antibody rat anti-mouse Mac2 marker of “inflammatory macrophages” (Cedarlane Laboratories, Burlington, NC), a polyclonal rabbit antibody to either human cleaved caspase-3 (Cell Signaling Technology, Boston, MA), vWf (Dako) or mouse C4d (developed in our laboratory , now available from Hycult, Biotech, Plymouth Meeting, PA).

    Techniques: Staining

    Measurements of apoptosis and C4d deposition The extent of apoptotic cells and nuclear fragments stained for activated caspase 3 was quantified in grids of 50 microns square superimposed on digital images captured on medium power. The number of squares containing apoptotic cells was much more widespread in cardiac grafts to FcγRIII-KO than WT recipients (A). Apoptosis was sparse in allografts to wild type recipients at the initial stages of rejection (6-8 days) and in the terminal phases of rejection (9-11 days). C4d deposition graded on the clinical criteria of distribution (focal versus diffuse) and intensity with each parameter scored on a 0 to 3+ scale demonstrated diffuse and intense deposits occurred more acutely in cardiac grafts to FcγRIII-KO than WT recipients (B). The more acute deposition of C4d correlated with more rapid generation of alloantibodies responses to cardiac grafts to FcγRIII-KO than WT recipients.

    Journal: Transplantation

    Article Title: The absence of FcγRIII results in increased pro-inflammatory response in FcγRIII-KO cardiac recipients

    doi: 10.1097/TP.0b013e31829c2455

    Figure Lengend Snippet: Measurements of apoptosis and C4d deposition The extent of apoptotic cells and nuclear fragments stained for activated caspase 3 was quantified in grids of 50 microns square superimposed on digital images captured on medium power. The number of squares containing apoptotic cells was much more widespread in cardiac grafts to FcγRIII-KO than WT recipients (A). Apoptosis was sparse in allografts to wild type recipients at the initial stages of rejection (6-8 days) and in the terminal phases of rejection (9-11 days). C4d deposition graded on the clinical criteria of distribution (focal versus diffuse) and intensity with each parameter scored on a 0 to 3+ scale demonstrated diffuse and intense deposits occurred more acutely in cardiac grafts to FcγRIII-KO than WT recipients (B). The more acute deposition of C4d correlated with more rapid generation of alloantibodies responses to cardiac grafts to FcγRIII-KO than WT recipients.

    Article Snippet: Slides were incubated for 30 minutes in a serum free blocking solution (Dako, Carpenteria, CA) followed by an overnight incubation with a monoclonal antibody rat anti-mouse Mac2 marker of “inflammatory macrophages” (Cedarlane Laboratories, Burlington, NC), a polyclonal rabbit antibody to either human cleaved caspase-3 (Cell Signaling Technology, Boston, MA), vWf (Dako) or mouse C4d (developed in our laboratory , now available from Hycult, Biotech, Plymouth Meeting, PA).

    Techniques: Staining

    Jasplakinolide (Jasplakinolide) attenuates isoflurane-mediated reduction in dendritic filopodial spines and enhancement of apoptosis in DIV4-7 neurons. Primary neurons (4-7 days in vitro – DIV4-7) were exposed to 1.4% isoflurane for 4 h with and without pre-treatment with the actin cytoskeleton stabilizer Jasplakinolide (1 h, 1 μM) and incubated with antibodies for drebrin (neuronal Factin binding protein) (A-C, quantitation shown in panel D), the apoptotic marker cleaved caspase 3 (cl-Csp3) (E-G, quantitation shown in panel 3H), and the nuclear marker DAPI. Pretreatment with Jasplakinolide significantly (C, n = 4-6; * p = 0.0144 vs . isoflurane) attenuated isoflurane-mediated decreased (B, # p = 0.0337 vs . basal) in drebrin immunofluorescence and significantly (n = 4-6; * p = 0.001 vs . isoflurane) decreased cl-Csp3 expression (G). Drebrin (green pixels) along dendrites is normalized to DAPI (blue pixels) and cl-Csp3 (red pixels) is normalized to DAPI. Scale bar, 10 μm. Error bars, standard error of the mean (s.e.m.).

    Journal: Anesthesiology

    Article Title: Isoflurane Neurotoxicity Is Mediated by p75NTR-RhoA Activation and Actin Depolymerization

    doi: 10.1097/ALN.0b013e318201dcb3

    Figure Lengend Snippet: Jasplakinolide (Jasplakinolide) attenuates isoflurane-mediated reduction in dendritic filopodial spines and enhancement of apoptosis in DIV4-7 neurons. Primary neurons (4-7 days in vitro – DIV4-7) were exposed to 1.4% isoflurane for 4 h with and without pre-treatment with the actin cytoskeleton stabilizer Jasplakinolide (1 h, 1 μM) and incubated with antibodies for drebrin (neuronal Factin binding protein) (A-C, quantitation shown in panel D), the apoptotic marker cleaved caspase 3 (cl-Csp3) (E-G, quantitation shown in panel 3H), and the nuclear marker DAPI. Pretreatment with Jasplakinolide significantly (C, n = 4-6; * p = 0.0144 vs . isoflurane) attenuated isoflurane-mediated decreased (B, # p = 0.0337 vs . basal) in drebrin immunofluorescence and significantly (n = 4-6; * p = 0.001 vs . isoflurane) decreased cl-Csp3 expression (G). Drebrin (green pixels) along dendrites is normalized to DAPI (blue pixels) and cl-Csp3 (red pixels) is normalized to DAPI. Scale bar, 10 μm. Error bars, standard error of the mean (s.e.m.).

    Article Snippet: Cleaved-caspase 3 (Cl-Csp3) (Cell Signaling, Danvers, MA) and drebrin (Abcam, Cambridge, MA) were used to detect apoptosis and to delineate the F-actin cytoskeleton respectively via immunofluorescence deconvolution microscopy.

    Techniques: In Vitro, Incubation, Binding Assay, Quantitation Assay, Marker, Immunofluorescence, Expressing

    Isoflurane exposure decreases neuritic processes and enhances neuronal apoptosis in DIV4-7 primary neurons. Primary neurons (4-7 days in vitro – DIV4-7) were exposed to 1.4% isoflurane for 2 h with and without pretreatment with TAT-Pep5 (15 min, 10 μM) and incubated with antibodies for drebrin (neuronal F-actin binding protein) (A-C, quantitation shown in panel D), the apoptotic marker, cleaved caspase 3 (cl-Csp3) (E-G, quantitation shown in panel H), and the nuclear marker DAPI. DIV4-7 neurons exposed to isoflurane exhibited a significant reduction (n = 4; # p = 0.005 vs . basal) in dendritic filopodial spines as indicated by decreased drebrin immunofluorescence along dendritic shafts (B) compared to control (Ctrl, A); isoflurane significantly enhanced cl-Csp-3 within the cell body (n = 5-7; # p = 0.04 vs . basal) (F) compared to Ctrl (E). Pretreatment with TAT-Pep5 significantly (n = 4) blocked the isoflurane-mediated decrease in drebrin (C, * p = 0.0348 vs . isoflurane) and the increase in cl-Csp3 (n = 5-7; * p = 0.031 vs . isoflurane) (G). Drebrin (green pixels) along dendrites is normalized to DAPI (blue pixels) or cl-Csp3 (red pixels) is normalized to DAPI. Scale bar, 10 μm. Error bars, standard error of the mean (s.e.m.).

    Journal: Anesthesiology

    Article Title: Isoflurane Neurotoxicity Is Mediated by p75NTR-RhoA Activation and Actin Depolymerization

    doi: 10.1097/ALN.0b013e318201dcb3

    Figure Lengend Snippet: Isoflurane exposure decreases neuritic processes and enhances neuronal apoptosis in DIV4-7 primary neurons. Primary neurons (4-7 days in vitro – DIV4-7) were exposed to 1.4% isoflurane for 2 h with and without pretreatment with TAT-Pep5 (15 min, 10 μM) and incubated with antibodies for drebrin (neuronal F-actin binding protein) (A-C, quantitation shown in panel D), the apoptotic marker, cleaved caspase 3 (cl-Csp3) (E-G, quantitation shown in panel H), and the nuclear marker DAPI. DIV4-7 neurons exposed to isoflurane exhibited a significant reduction (n = 4; # p = 0.005 vs . basal) in dendritic filopodial spines as indicated by decreased drebrin immunofluorescence along dendritic shafts (B) compared to control (Ctrl, A); isoflurane significantly enhanced cl-Csp-3 within the cell body (n = 5-7; # p = 0.04 vs . basal) (F) compared to Ctrl (E). Pretreatment with TAT-Pep5 significantly (n = 4) blocked the isoflurane-mediated decrease in drebrin (C, * p = 0.0348 vs . isoflurane) and the increase in cl-Csp3 (n = 5-7; * p = 0.031 vs . isoflurane) (G). Drebrin (green pixels) along dendrites is normalized to DAPI (blue pixels) or cl-Csp3 (red pixels) is normalized to DAPI. Scale bar, 10 μm. Error bars, standard error of the mean (s.e.m.).

    Article Snippet: Cleaved-caspase 3 (Cl-Csp3) (Cell Signaling, Danvers, MA) and drebrin (Abcam, Cambridge, MA) were used to detect apoptosis and to delineate the F-actin cytoskeleton respectively via immunofluorescence deconvolution microscopy.

    Techniques: In Vitro, Incubation, Binding Assay, Quantitation Assay, Marker, Immunofluorescence

    Characterization of 32 P uptake by the cell. A. 32 P is directly incorporated into cellular DNA. Mouse CRL2836 or human HeLa S3 cell lines were incubated overnight with 32 P[PO 4 ] and then grown for 48 h in non-radioactive medium (lanes 1 through 4), or grown for 24 h in non-radioactive medium, grown for 24 h with 32 P[PO 4 ], and then grown for 24 h in non-radioactive medium (lanes 5 through 8), or grown for 48 h in non-radioactive medium, then grown for 24 h with 32 P[PO 4 ] (lanes 9 through 12). The extracted nucleic acids were incubated with DNase I, the digestion products were run on a 5% polyacrylamide gel and exposed to film. B. Apoptosis induced by 32 P in mouse CRL2836 cells. Mouse CRL2836 cells were incubated with 0, 2.5, 5, 10 or 20 μCi 32 P[PO 4 ] for 24 h, and non-radioactive medium added for an additional 24 h. Protein was extracted from each well and analyzed for apoptosis by western blots using antibody to cleaved caspase-3 protein (Lanes 1 through 5). Antibody against beta-actin was used to verify identical amounts of protein were loaded (lanes 6 through 10).

    Journal: PLoS ONE

    Article Title: Phosphorus-32, a Clinically Available Drug, Inhibits Cancer Growth by Inducing DNA Double-Strand Breakage

    doi: 10.1371/journal.pone.0128152

    Figure Lengend Snippet: Characterization of 32 P uptake by the cell. A. 32 P is directly incorporated into cellular DNA. Mouse CRL2836 or human HeLa S3 cell lines were incubated overnight with 32 P[PO 4 ] and then grown for 48 h in non-radioactive medium (lanes 1 through 4), or grown for 24 h in non-radioactive medium, grown for 24 h with 32 P[PO 4 ], and then grown for 24 h in non-radioactive medium (lanes 5 through 8), or grown for 48 h in non-radioactive medium, then grown for 24 h with 32 P[PO 4 ] (lanes 9 through 12). The extracted nucleic acids were incubated with DNase I, the digestion products were run on a 5% polyacrylamide gel and exposed to film. B. Apoptosis induced by 32 P in mouse CRL2836 cells. Mouse CRL2836 cells were incubated with 0, 2.5, 5, 10 or 20 μCi 32 P[PO 4 ] for 24 h, and non-radioactive medium added for an additional 24 h. Protein was extracted from each well and analyzed for apoptosis by western blots using antibody to cleaved caspase-3 protein (Lanes 1 through 5). Antibody against beta-actin was used to verify identical amounts of protein were loaded (lanes 6 through 10).

    Article Snippet: A western blot using a primary antibody to cleaved caspase-3 protein (Cell Signaling Technology, Boston, MA) was used to assay for apoptosis.

    Techniques: Incubation, Western Blot

    Combination of oncolytic herpes simplex virus (oHSV) and S-TRAIL leads to caspase-3/7-mediated apoptosis in resistant glioblastoma multiforme (GBM) cells . ( a – d ) Caspase-3/7 activity and cell viability of ( a , c ) LN229 and ( b , d ) GBM8F treated with

    Journal: Molecular Therapy

    Article Title: Multimechanistic Tumor Targeted Oncolytic Virus Overcomes Resistance in Brain Tumors

    doi: 10.1038/mt.2012.175

    Figure Lengend Snippet: Combination of oncolytic herpes simplex virus (oHSV) and S-TRAIL leads to caspase-3/7-mediated apoptosis in resistant glioblastoma multiforme (GBM) cells . ( a – d ) Caspase-3/7 activity and cell viability of ( a , c ) LN229 and ( b , d ) GBM8F treated with

    Article Snippet: For cleaved-caspase-3 staining, sections were incubated for 1 hour in a blocking solution (0.3% bovine serum albumin, 8% goat serum, and 0.3% Triton-X100) at room temperature, followed by incubation at 4 °C overnight with anti-cleaved-caspase-3 (Cell Signaling) diluted in blocking solution.

    Techniques: Activity Assay

    Transplanted ferumoxytol‐labeled human neural progenitor cells differentiate in the porcine spinal cord. Representative fluorescent micrographs of immunohistochemical staining with a mouse monoclonal anti‐human glial fibrillary acidic protein (GFAP) antibody (green) and a rabbit polyclonal anti‐human nestin antibody (red) in unlabeled human neural progenitor cells (hNPC) (A) , ferumoxytol‐labeled hNPC‐(F) Low (B) , and hNPC‐F High (C) grafts from postoperative day 105 are shown with high‐magnification insets. All nuclei are observed with 49,6‐diamidino‐2‐phenylindole staining (blue). Five grafts from each condition with > 10% engraftment were chosen for analysis. GFAP+ or nestin+ signal was not observed on the contralateral side or in rejected grafts. High‐magnification images of unlabeled hNPC ( D ), hNPC‐F Low ( E ), and hNPC‐F High ( F ) cell grafts expressing human nucleus (HuNu+) (green) and Ki67+ (red) show few proliferating transplanted cells (solid arrows). High‐magnification images of unlabeled hNPC (G) , hNPC‐F Low (H) , and hNPC‐F High (I) cell grafts expressing HuNu+ (green) and cleaved caspase‐3 (CC3) (red) show few apoptotic transplanted cells (open arrows). The relative expression of GFAP+ and nestin+ was calculated for all groups (J‐L) . The relative expression of HuNu+ and Ki67+ was calculated (M) , as was that between HuNu+ and CC3 (N) . Scale bars = ×4, 500 μm (A‐C) ; ×100, 10 μm (A‐C) (insets); ×40, 25 μm (D‐I) . Graphs are displayed as mean ± SD. Abbreviations: CC3, cleaved caspase 3; DAPI, 4′,6‐diamidino‐2‐phenylindole; F, ferumoxytol; GFAP, glial fibrillary acidic protein; hNPC, human neural progenitor cell; HuNu, human nucleus; ns, not significant.

    Journal: Stem Cells Translational Medicine

    Article Title: Ferumoxytol Labeling of Human Neural Progenitor Cells for Diagnostic Cellular Tracking in the Porcine Spinal Cord with Magnetic Resonance Imaging

    doi: 10.5966/sctm.2015-0422

    Figure Lengend Snippet: Transplanted ferumoxytol‐labeled human neural progenitor cells differentiate in the porcine spinal cord. Representative fluorescent micrographs of immunohistochemical staining with a mouse monoclonal anti‐human glial fibrillary acidic protein (GFAP) antibody (green) and a rabbit polyclonal anti‐human nestin antibody (red) in unlabeled human neural progenitor cells (hNPC) (A) , ferumoxytol‐labeled hNPC‐(F) Low (B) , and hNPC‐F High (C) grafts from postoperative day 105 are shown with high‐magnification insets. All nuclei are observed with 49,6‐diamidino‐2‐phenylindole staining (blue). Five grafts from each condition with > 10% engraftment were chosen for analysis. GFAP+ or nestin+ signal was not observed on the contralateral side or in rejected grafts. High‐magnification images of unlabeled hNPC ( D ), hNPC‐F Low ( E ), and hNPC‐F High ( F ) cell grafts expressing human nucleus (HuNu+) (green) and Ki67+ (red) show few proliferating transplanted cells (solid arrows). High‐magnification images of unlabeled hNPC (G) , hNPC‐F Low (H) , and hNPC‐F High (I) cell grafts expressing HuNu+ (green) and cleaved caspase‐3 (CC3) (red) show few apoptotic transplanted cells (open arrows). The relative expression of GFAP+ and nestin+ was calculated for all groups (J‐L) . The relative expression of HuNu+ and Ki67+ was calculated (M) , as was that between HuNu+ and CC3 (N) . Scale bars = ×4, 500 μm (A‐C) ; ×100, 10 μm (A‐C) (insets); ×40, 25 μm (D‐I) . Graphs are displayed as mean ± SD. Abbreviations: CC3, cleaved caspase 3; DAPI, 4′,6‐diamidino‐2‐phenylindole; F, ferumoxytol; GFAP, glial fibrillary acidic protein; hNPC, human neural progenitor cell; HuNu, human nucleus; ns, not significant.

    Article Snippet: Proliferation was assessed with HuNu/Ki67 (ab15580; Abcam; 1/500) costain and apoptosis with HuNu/Cleaved Caspase 3 (9661; Cell Signaling Technology, Danvers, MA, https://www.cellsignal.com ; 1/125) costain.

    Techniques: Labeling, Immunohistochemistry, Staining, Expressing

    Effects of delayed isoflurane preconditioning on the expression of eNOS (A), iNOS(B), COX-2 (C), Cleaved Caspase-3 (D) in rat hearts exposed to ischemia-reperfusion (IR). IR: ischemia reperfusion; Iso: isoflurane; TAC: Transverse aortic constriction. Data are mean ± SD, n = 6 hearts/group. *P

    Journal: Scientific Reports

    Article Title: Ventricular hypertrophy blocked delayed anesthetic cardioprotection in rats by alteration of iNOS/COX-2 signaling

    doi: 10.1038/srep07071

    Figure Lengend Snippet: Effects of delayed isoflurane preconditioning on the expression of eNOS (A), iNOS(B), COX-2 (C), Cleaved Caspase-3 (D) in rat hearts exposed to ischemia-reperfusion (IR). IR: ischemia reperfusion; Iso: isoflurane; TAC: Transverse aortic constriction. Data are mean ± SD, n = 6 hearts/group. *P

    Article Snippet: The expression of myocardial Cleaved Caspase-3 (Cell Signaling Technology, Beverly, MA, USA) was determined by immunoblotting .

    Techniques: Expressing

    ) (A) Immunoblotting of spinal cord lysates from 4 SMAΔ7 mice and 3 wild-type mice at PND12. Spinal cords from SMAΔ7 mice show low expression of the full-length SMN protein, and higher levels of UPR markers cleaved Atf6 and Chop. (B) GUA treatment results in reduction of Ire1 , Chop , Atf4 and Pfdn5 mRNA expression in spinal cords of SMAΔ7 mice, to levels similar in control wild-type mice. (C) Decreased Caspase-3 expression, as well as ER stress-associated Caspase-4 and Caspase-12 expression was observed in spinal cord lysates of SMAΔ7 mice treated with GUA. (D) While mRNA levels of Smn remain low in the SMAΔ7 animals, expression of the MN marker ChAT increases in the GUA-treated animals. (E) Western blot analysis of spinal cord lysates of SMAΔ7 and wild-type mice, treated with either DMSO or GUA, showing that GUA reduced UPR marker expression but led to increased MN markers Smi-32 and ChAT. (F) Representative images of ventral horn ChAT + MNs (red) of the respectively treated animals. Scale bar represents 100 μm. (G) Quantification of the number of ChAT + MNs in the ventral horns of each 10 μm section. 4–6 animals from each condition, and 20 lumbar sections representing the entire lumbar spinal cord were analyzed. Error bars are mean ± S.E.M. (H) Measurement of soma sizes of ChAT + ventral horn MNs indicating that the average MN size of SMAΔ7 animals treated with GUA is significantly larger than DMSO-treated SMAΔ7 animals. (I) Kaplan-Meier survival curve of SMAΔ7 mice treated with either DMSO (n = 9) or GUA (n = 8). Survival increased by approximately 35% (4 days) upon GUA treatment ( p = 0.0122). (J) Righting reflex time of DMSO and GUA-treated mutant and wild-type animals indicating that GUA-treated SMAΔ7 mice have significantly reduced righting reflex compared to DMSO controls ( p = 0.033). (K) Neuromuscular junctions (NMJs) of hindlimb muscles stained with α-bungarotoxin (BTX) and neurofilaments are identified by Smi-32 staining. Denervated NMJs are evident in DMSO-treated SMAΔ7 mice whereas the same region in GUA-treated SMAΔ7 animals showed preservation of NMJ innervation, similar to a wild-type animal. Magnified images of the white boxes are shown in the bottom panel. Scale bars represent 100 μm. (L) Analysis of innervation status of hindlimb NMJs in respective treated mice. NMJs are scored as innervated when there is Smi-32 staining of neurofilaments at the NMJs. GUA treatment in SMA mice led to a 20% increase in innervated NMJs ( p = 0.0125).

    Journal: Cell stem cell

    Article Title: Genome-Wide RNA-Seq of Human Motor Neurons Implicates Selective ER Stress Activation in Spinal Muscular Atrophy

    doi: 10.1016/j.stem.2015.08.003

    Figure Lengend Snippet: ) (A) Immunoblotting of spinal cord lysates from 4 SMAΔ7 mice and 3 wild-type mice at PND12. Spinal cords from SMAΔ7 mice show low expression of the full-length SMN protein, and higher levels of UPR markers cleaved Atf6 and Chop. (B) GUA treatment results in reduction of Ire1 , Chop , Atf4 and Pfdn5 mRNA expression in spinal cords of SMAΔ7 mice, to levels similar in control wild-type mice. (C) Decreased Caspase-3 expression, as well as ER stress-associated Caspase-4 and Caspase-12 expression was observed in spinal cord lysates of SMAΔ7 mice treated with GUA. (D) While mRNA levels of Smn remain low in the SMAΔ7 animals, expression of the MN marker ChAT increases in the GUA-treated animals. (E) Western blot analysis of spinal cord lysates of SMAΔ7 and wild-type mice, treated with either DMSO or GUA, showing that GUA reduced UPR marker expression but led to increased MN markers Smi-32 and ChAT. (F) Representative images of ventral horn ChAT + MNs (red) of the respectively treated animals. Scale bar represents 100 μm. (G) Quantification of the number of ChAT + MNs in the ventral horns of each 10 μm section. 4–6 animals from each condition, and 20 lumbar sections representing the entire lumbar spinal cord were analyzed. Error bars are mean ± S.E.M. (H) Measurement of soma sizes of ChAT + ventral horn MNs indicating that the average MN size of SMAΔ7 animals treated with GUA is significantly larger than DMSO-treated SMAΔ7 animals. (I) Kaplan-Meier survival curve of SMAΔ7 mice treated with either DMSO (n = 9) or GUA (n = 8). Survival increased by approximately 35% (4 days) upon GUA treatment ( p = 0.0122). (J) Righting reflex time of DMSO and GUA-treated mutant and wild-type animals indicating that GUA-treated SMAΔ7 mice have significantly reduced righting reflex compared to DMSO controls ( p = 0.033). (K) Neuromuscular junctions (NMJs) of hindlimb muscles stained with α-bungarotoxin (BTX) and neurofilaments are identified by Smi-32 staining. Denervated NMJs are evident in DMSO-treated SMAΔ7 mice whereas the same region in GUA-treated SMAΔ7 animals showed preservation of NMJ innervation, similar to a wild-type animal. Magnified images of the white boxes are shown in the bottom panel. Scale bars represent 100 μm. (L) Analysis of innervation status of hindlimb NMJs in respective treated mice. NMJs are scored as innervated when there is Smi-32 staining of neurofilaments at the NMJs. GUA treatment in SMA mice led to a 20% increase in innervated NMJs ( p = 0.0125).

    Article Snippet: Primary antibodies used in this study (and their respective dilutions) are as follow: rabbit ISL1 (Abcam ab109517; 1:1000), mouse TUJ1 (Covance MMS-435P; 1:1000), mouse HB9 (DSHB 81.5C10; 1:200), mouse ISL1 (DSHB 39.4D5), mouse SMI-32 (Calbiochem NE-1023; 1:1000), goat ChAT (Millipore AB114P; 1:100), rabbit ATF6 (Abcam ab37149; 1:500), rabbit ATF4 (Cell Signaling #11815; 1:100), rabbit cleaved Caspase-3 (Cell Signaling #9661) and mouse monoclonal SMN (BD Pharmingen; 1:250).

    Techniques: Mouse Assay, Expressing, Marker, Western Blot, Mutagenesis, Staining, Preserving

    ) (A) Quantitative PCR of FACS-purified HB9 + MNs derived from wild-type and SMA cultures at day 31. SMA MNs from 1-38G and 1-51N show higher expression of ER stress markers. Only 1-38G MNs show increased expression of markers characteristic of chronic ER stress: PERK , CHOP and CASP3 . Gene expression is normalized to GAPDH . (B) Co-staining of MN marker ISL1 (green) and ER stress marker ATF6 (red) in wild-type and SMA MN cultures. Scale bar indicates 25 μm. Nuclear ATF6 intensities of ISL1 + and ISL1 − cells are also measured. (C) Co-staining of motor neuron marker ISL1 (green) and ER stress marker ATF4 (red) in wild-type and SMA MN cultures. Scale bar indicates 100 μm. The graph depicts percentage of ATF4 + cells in whole cultures, as well as percentage of ISL1 + MNs co-expressing ATF4. (D–E) Quantification of ISL1 + MNs co-expressing ATF4 and cCASP3 respectively at days 23, 28 and 31, showing increasing ER stress over time, co-incident with increased apoptosis in SMA MNs. (F) Immunostaining analysis indicating that majority of MNs undergoing ER stress (ISL1 + ATF4 + MNs) also co-express ChAT. (G–H) qPCR and western blot analyses respectively show that knockdown of SMN in wild-type BJ-riPS MNs increased UPR target gene expression in a dose-dependent manner. (I) Co-staining of ISL1 (green) and ATF6 (red) in wild-type MNs transfected with non-targeting siRNA (si-NT) and SMN siRNA (si-SMN) at the indicated dose. The scale bar indicates 50 μm. Increase in nuclear ATF6 intensity upon SMN knockdown is graphically represented. (J) Co-staining of ISL1 (green) and ATF4 (red) in SMN knockdown BJ-riPS cultures. Scale bar indicates 100 μm. The graph shows increase in percentage of total cells and ISL1 + MNs co-expressing ATF4 in SMN knockdown conditions.

    Journal: Cell stem cell

    Article Title: Genome-Wide RNA-Seq of Human Motor Neurons Implicates Selective ER Stress Activation in Spinal Muscular Atrophy

    doi: 10.1016/j.stem.2015.08.003

    Figure Lengend Snippet: ) (A) Quantitative PCR of FACS-purified HB9 + MNs derived from wild-type and SMA cultures at day 31. SMA MNs from 1-38G and 1-51N show higher expression of ER stress markers. Only 1-38G MNs show increased expression of markers characteristic of chronic ER stress: PERK , CHOP and CASP3 . Gene expression is normalized to GAPDH . (B) Co-staining of MN marker ISL1 (green) and ER stress marker ATF6 (red) in wild-type and SMA MN cultures. Scale bar indicates 25 μm. Nuclear ATF6 intensities of ISL1 + and ISL1 − cells are also measured. (C) Co-staining of motor neuron marker ISL1 (green) and ER stress marker ATF4 (red) in wild-type and SMA MN cultures. Scale bar indicates 100 μm. The graph depicts percentage of ATF4 + cells in whole cultures, as well as percentage of ISL1 + MNs co-expressing ATF4. (D–E) Quantification of ISL1 + MNs co-expressing ATF4 and cCASP3 respectively at days 23, 28 and 31, showing increasing ER stress over time, co-incident with increased apoptosis in SMA MNs. (F) Immunostaining analysis indicating that majority of MNs undergoing ER stress (ISL1 + ATF4 + MNs) also co-express ChAT. (G–H) qPCR and western blot analyses respectively show that knockdown of SMN in wild-type BJ-riPS MNs increased UPR target gene expression in a dose-dependent manner. (I) Co-staining of ISL1 (green) and ATF6 (red) in wild-type MNs transfected with non-targeting siRNA (si-NT) and SMN siRNA (si-SMN) at the indicated dose. The scale bar indicates 50 μm. Increase in nuclear ATF6 intensity upon SMN knockdown is graphically represented. (J) Co-staining of ISL1 (green) and ATF4 (red) in SMN knockdown BJ-riPS cultures. Scale bar indicates 100 μm. The graph shows increase in percentage of total cells and ISL1 + MNs co-expressing ATF4 in SMN knockdown conditions.

    Article Snippet: Primary antibodies used in this study (and their respective dilutions) are as follow: rabbit ISL1 (Abcam ab109517; 1:1000), mouse TUJ1 (Covance MMS-435P; 1:1000), mouse HB9 (DSHB 81.5C10; 1:200), mouse ISL1 (DSHB 39.4D5), mouse SMI-32 (Calbiochem NE-1023; 1:1000), goat ChAT (Millipore AB114P; 1:100), rabbit ATF6 (Abcam ab37149; 1:500), rabbit ATF4 (Cell Signaling #11815; 1:100), rabbit cleaved Caspase-3 (Cell Signaling #9661) and mouse monoclonal SMN (BD Pharmingen; 1:250).

    Techniques: Real-time Polymerase Chain Reaction, FACS, Purification, Derivative Assay, Expressing, Staining, Marker, Immunostaining, Western Blot, Transfection

    (a, b) Western blotting for the ratio of phosphorylated insulin receptor on tyrosine 1150/1151 (p-IR) and IRS-1 Ser307 (p-IRS-1) to total protein. (c) Western blotting for the ratio of Akt2 to β -actin. (d) ELISA results for cleaved caspase 3 levels. ∗ P

    Journal: Mediators of Inflammation

    Article Title: Epac1 Restores Normal Insulin Signaling through a Reduction in Inflammatory Cytokines

    doi: 10.1155/2018/3809092

    Figure Lengend Snippet: (a, b) Western blotting for the ratio of phosphorylated insulin receptor on tyrosine 1150/1151 (p-IR) and IRS-1 Ser307 (p-IRS-1) to total protein. (c) Western blotting for the ratio of Akt2 to β -actin. (d) ELISA results for cleaved caspase 3 levels. ∗ P

    Article Snippet: ELISA A cleaved caspase 3 ELISA (Cell Signaling Corp, Danvers, MA) was done according to the manufacturer's instructions on both mouse whole retinal lysates and REC.

    Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

    (a) Western blotting for the ratio of IL-1 β siRNA to β -actin in REC grown in normal glucose (NG) and high glucose (HG). Some REC grown in HG were transfected with IL-1 β siRNA or scrambled siRNA (sc). (b–d) Western blotting for the ratio of phosphorylated insulin receptor on tyrosine 1150/1151 (p-IR), IRS-1 Ser307 (p-IRS-1), and Akt (p-Akt) to total protein. (e) ELISA results for cleaved caspase 3 levels. ∗ P

    Journal: Mediators of Inflammation

    Article Title: Epac1 Restores Normal Insulin Signaling through a Reduction in Inflammatory Cytokines

    doi: 10.1155/2018/3809092

    Figure Lengend Snippet: (a) Western blotting for the ratio of IL-1 β siRNA to β -actin in REC grown in normal glucose (NG) and high glucose (HG). Some REC grown in HG were transfected with IL-1 β siRNA or scrambled siRNA (sc). (b–d) Western blotting for the ratio of phosphorylated insulin receptor on tyrosine 1150/1151 (p-IR), IRS-1 Ser307 (p-IRS-1), and Akt (p-Akt) to total protein. (e) ELISA results for cleaved caspase 3 levels. ∗ P

    Article Snippet: ELISA A cleaved caspase 3 ELISA (Cell Signaling Corp, Danvers, MA) was done according to the manufacturer's instructions on both mouse whole retinal lysates and REC.

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

    Western blotting for the ratio of phosphorylated insulin receptor on tyrosine 1150/1151 (p-IR) (a), IRS-1 Ser307 (p-IRS-1) (b), and Akt (p-Akt) (c) to total protein in whole retinal lysates from Epac1 floxed mice or Epac1 Cdh5 Cre-lox mice. ELISA results for cleaved caspase 3 (d). ∗ P

    Journal: Mediators of Inflammation

    Article Title: Epac1 Restores Normal Insulin Signaling through a Reduction in Inflammatory Cytokines

    doi: 10.1155/2018/3809092

    Figure Lengend Snippet: Western blotting for the ratio of phosphorylated insulin receptor on tyrosine 1150/1151 (p-IR) (a), IRS-1 Ser307 (p-IRS-1) (b), and Akt (p-Akt) (c) to total protein in whole retinal lysates from Epac1 floxed mice or Epac1 Cdh5 Cre-lox mice. ELISA results for cleaved caspase 3 (d). ∗ P

    Article Snippet: ELISA A cleaved caspase 3 ELISA (Cell Signaling Corp, Danvers, MA) was done according to the manufacturer's instructions on both mouse whole retinal lysates and REC.

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

    (a) Western blotting for the ratio of TNF α siRNA to β -actin in REC grown in normal glucose (NG) and high glucose (HG). Some REC grown in HG were transfected with TNF α siRNA or scrambled siRNA (sc). (b–d) Western blotting for the ratio of phosphorylated insulin receptor on tyrosine 1150/1151 (p-IR), IRS-1 Ser307 (p-IRS-1), and Akt (p-Akt) to total protein. (e) ELISA results for cleaved caspase 3 levels. ∗ P

    Journal: Mediators of Inflammation

    Article Title: Epac1 Restores Normal Insulin Signaling through a Reduction in Inflammatory Cytokines

    doi: 10.1155/2018/3809092

    Figure Lengend Snippet: (a) Western blotting for the ratio of TNF α siRNA to β -actin in REC grown in normal glucose (NG) and high glucose (HG). Some REC grown in HG were transfected with TNF α siRNA or scrambled siRNA (sc). (b–d) Western blotting for the ratio of phosphorylated insulin receptor on tyrosine 1150/1151 (p-IR), IRS-1 Ser307 (p-IRS-1), and Akt (p-Akt) to total protein. (e) ELISA results for cleaved caspase 3 levels. ∗ P

    Article Snippet: ELISA A cleaved caspase 3 ELISA (Cell Signaling Corp, Danvers, MA) was done according to the manufacturer's instructions on both mouse whole retinal lysates and REC.

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

    Therapeutic RDV but not LPV/RTV-IFNb diminishes signs of ALI. a Representative images of the histological features of acute lung injury 6 dpi comparing a mock-infected mouse to the therapeutic treatment groups described in Figs. 5 and 6 . Symbols identifying example features of disease are indicated in the figure. b American Thoracic Society Lung Injury Score derived as described in Fig. 3 . The numbers of animals per group quantitated: vehicle RDV N = 7, RDV N = 7, vehicle LPV/RTV-IFNb N = 9, LPV/RTV-IFNb low N = 7, LPV/RTV-IFNb high N = 8. c Diffuse alveolar damage score quantitating the degree of cellular sloughing, necrosis, and breakdown of barrier epithelium and vascular leakage. For both b and c , scores were blindly assessed in three random high power (×60) fields of diseased lung tissue sections. d Quantitation of cleaved caspase-3 antigen staining in lung tissue sections from studies described in Figs. 5 –7. Cleaved caspase-3 is a marker of cell death. The numbers of animals per group quantitated for all groups was N = 5/group. For the box and whisker plots, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range. For b – d , asterisks indicate statistical significance by one-way ANOVA and Kruskal–Wallis multiple comparison test.

    Journal: Nature Communications

    Article Title: Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against MERS-CoV

    doi: 10.1038/s41467-019-13940-6

    Figure Lengend Snippet: Therapeutic RDV but not LPV/RTV-IFNb diminishes signs of ALI. a Representative images of the histological features of acute lung injury 6 dpi comparing a mock-infected mouse to the therapeutic treatment groups described in Figs. 5 and 6 . Symbols identifying example features of disease are indicated in the figure. b American Thoracic Society Lung Injury Score derived as described in Fig. 3 . The numbers of animals per group quantitated: vehicle RDV N = 7, RDV N = 7, vehicle LPV/RTV-IFNb N = 9, LPV/RTV-IFNb low N = 7, LPV/RTV-IFNb high N = 8. c Diffuse alveolar damage score quantitating the degree of cellular sloughing, necrosis, and breakdown of barrier epithelium and vascular leakage. For both b and c , scores were blindly assessed in three random high power (×60) fields of diseased lung tissue sections. d Quantitation of cleaved caspase-3 antigen staining in lung tissue sections from studies described in Figs. 5 –7. Cleaved caspase-3 is a marker of cell death. The numbers of animals per group quantitated for all groups was N = 5/group. For the box and whisker plots, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range. For b – d , asterisks indicate statistical significance by one-way ANOVA and Kruskal–Wallis multiple comparison test.

    Article Snippet: Lung tissue sections were stained for cleaved caspase-3 antigen (1:500, Cell Signaling #9664) by the Animal Histopathology & Laboratory Medicine Core at UNC.

    Techniques: Infection, Derivative Assay, Quantitation Assay, Staining, Marker, Whisker Assay

    Substitution of Matrigel/rCOL by hCOL from fibroblasts leads to lumenized EC network formation with involvement of apoptosis. ( a , b ) 3D hydrogel constructs containing GFP positive HUVECs and hASCs and hCOL after 14 days of cultivation in SFM. ( c ) Virtual stacks of GFP-HUVECs co-cultured with hASCs in hCOL for 14 days and stained for α-SMA. Nuclei were counterstained with DAPI. α-SMA positive cells are in physical contact with GFP-HUVECs. ( d ) Virtual stacks of HUVECs co-cultured with hASCs and incubated with Texas red-labeled Dextran. ( e – e ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM and incubated with Texas red-labeled dextran. Cryo-sections were stained with DAPI. ( e ) GFP-HUVEC, ( e ’) DAPI, ( e ”) Texas red-labeled dextran, ( e ”’) Superimposed image of ( e , e ’, e ”). Intense DAPI staining of fragmented DNA is localized within the lumen filled with Texas red-labeled dextran. ( f – f ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM. Cryo-sections were stained for cleaved caspase 3 and DAPI. ( f ) GFP-HUVEC, ( f ’) DAPI, ( f ”) cleaved caspase 3 (CASP3), ( f ”’) superimposed image of ( f , f ’, f ”). Intense DAPI staining of fragmented DNA is localized within the lumen and co-localizes with the signal for cleaved caspase 3. Scale bar: ( a ) 500 µm; ( b ) 200 µm; ( c – f ”’) 30 µm.

    Journal: Scientific Reports

    Article Title: Formation of three-dimensional tubular endothelial cell networks under defined serum-free cell culture conditions in human collagen hydrogels

    doi: 10.1038/s41598-019-41985-6

    Figure Lengend Snippet: Substitution of Matrigel/rCOL by hCOL from fibroblasts leads to lumenized EC network formation with involvement of apoptosis. ( a , b ) 3D hydrogel constructs containing GFP positive HUVECs and hASCs and hCOL after 14 days of cultivation in SFM. ( c ) Virtual stacks of GFP-HUVECs co-cultured with hASCs in hCOL for 14 days and stained for α-SMA. Nuclei were counterstained with DAPI. α-SMA positive cells are in physical contact with GFP-HUVECs. ( d ) Virtual stacks of HUVECs co-cultured with hASCs and incubated with Texas red-labeled Dextran. ( e – e ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM and incubated with Texas red-labeled dextran. Cryo-sections were stained with DAPI. ( e ) GFP-HUVEC, ( e ’) DAPI, ( e ”) Texas red-labeled dextran, ( e ”’) Superimposed image of ( e , e ’, e ”). Intense DAPI staining of fragmented DNA is localized within the lumen filled with Texas red-labeled dextran. ( f – f ”’) Images of cryo-sections from constructs containing GFP-HUVECs, hASCs and hCOL. Constructs were cultivated for 14 days in SFM. Cryo-sections were stained for cleaved caspase 3 and DAPI. ( f ) GFP-HUVEC, ( f ’) DAPI, ( f ”) cleaved caspase 3 (CASP3), ( f ”’) superimposed image of ( f , f ’, f ”). Intense DAPI staining of fragmented DNA is localized within the lumen and co-localizes with the signal for cleaved caspase 3. Scale bar: ( a ) 500 µm; ( b ) 200 µm; ( c – f ”’) 30 µm.

    Article Snippet: Blocking was performed with 2% donkey serum in PBS for 20 minutes at room temperature followed by overnight incubation with primary antibody α-smooth muscle actin (α-SMA) (DAKO, 1:400) or cleaved caspase-3 (CASP3) (Cell Signaling Technology, 1:200) at 4 °C.

    Techniques: Construct, Cell Culture, Staining, Incubation, Labeling