apoptosis Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher dead cell apoptosis kit
    Dead Cell Apoptosis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dead cell apoptosis kit/product/Thermo Fisher
    Average 99 stars, based on 3229 article reviews
    Price from $9.99 to $1999.99
    dead cell apoptosis kit - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    99
    Millipore situ apoptosis detection kit
    Concomitant loss of MLKL and Caspase-8, but not loss of RIPK1 kinase activity or combined loss of RIPK3 and Caspase-8, promotes survival of LUBAC-deficient mice a, Representative images of E10.5 ( n =6 embryos/genotype), scale bar: 2 mm, E14.5 ( n =12 Ripk1 K45A Hoil-1 +/- , n =5 Ripk1 K45A Hoil-1 -/- embryos/genotype) and E15.5 embryos ( n =3 embryos/genotype). Scale bar: 5 mm. *: poor yolk sac vascularisation. b, f, Representative images of yolk sac vascularisation (PECAM-1, red) and <t>apoptosis</t> (cleaved (cl.) Caspase-3, green) at E14.5 (b) or E13.5 (f) and quantification. Mean ± s.e.m. and P values from unpaired two-tailed t -tests (b) or one-way ANOVA (f) are shown. Scale bar: 50 µm. c, FADD-IP in MEFs treated for 3 h with zVAD-fmk ± TNF ( n . d, Cell death by PI incorporation in MEFs ± TNF (10 ng/ml) or LT-α. Mean ± s.e.m. ( n =3 independent experiments) and P values (**** P
    Situ Apoptosis Detection Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/situ apoptosis detection kit/product/Millipore
    Average 99 stars, based on 10129 article reviews
    Price from $9.99 to $1999.99
    situ apoptosis detection kit - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    92
    Becton Dickinson apoptosis
    MPA but not NET-A or P4 increases Vpr-mediated <t>apoptosis</t> in a dose-dependent manner. (A) PBMCs were treated with or without 5 µM Vpr peptide (amino acids 52–96) and increasing concentrations of MPA, NET-A or P4 for 24 hrs. The graph shows results pooled from two independent experiments with samples from three donors. (B) PBMCs were treated with 100 nM MPA, 10 µM NET-A, 1 µM P4 or in combination with 5 µM Vpr peptide (amino acids 52–96) for 24 hrs. Cells were stained and acquired by flow cytometry as described in the materials and methods . A tryptic BSA digest served as a control wherever Vpr peptide was not added, as for results in figure 4 . The histogram shows results pooled from two independent experiments with samples from three different donors compared to those in figure A. donors. Statistical trend analysis for panel A was performed by the Wilcox rank-sum test, as further extended by Cuzick [85] , and showed a significant trend only for MPA plus Vpr (p = 0.012) and P4 minus Vpr (p = 0.012). Statistical significance in panel B was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p
    Apoptosis, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 38464 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apoptosis/product/Becton Dickinson
    Average 92 stars, based on 38464 article reviews
    Price from $9.99 to $1999.99
    apoptosis - by Bioz Stars, 2020-11
    92/100 stars
      Buy from Supplier

    94
    Becton Dickinson fitc annexin v apoptosis detection kit
    The reverse signaling of tmTNF-α promotes AICD through upregulating FasL/Fas and TRAIL/DR4 (A, B, C, D) Jurkat cells were simultaneously stimulated with PHA-P (5 μg/ml) and TNF-α pAb (1:100) for 24 h. PHA-preactivated primary T cells were restimulated with αCD3 (10 μg/ml) and TNF-α pAb for 24 h. Expression of FasL/Fas (A, B) and TRAIL/DR4/DR5 (C, D) was detected by flow cytometry. (E, F) Jurkat cells transfected with 100 nM siRNA for FasL or TRAIL for 48 h were activated with PHA-P (5 μg/ml) in the absence or presence of TNF-α pAb for 24 h. The mRNA levels of FasL and TRAIL were determined by Real-time qPCR (E) and the <t>apoptosis</t> was detected by Annexin V/PI (F). All the quantitative data represent means ± S.D. of at least three independent experiments. * p
    Fitc Annexin V Apoptosis Detection Kit, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 6483 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fitc annexin v apoptosis detection kit/product/Becton Dickinson
    Average 94 stars, based on 6483 article reviews
    Price from $9.99 to $1999.99
    fitc annexin v apoptosis detection kit - by Bioz Stars, 2020-11
    94/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc apoptosis
    The reverse signaling of tmTNF-α promotes AICD through upregulating FasL/Fas and TRAIL/DR4 (A, B, C, D) Jurkat cells were simultaneously stimulated with PHA-P (5 μg/ml) and TNF-α pAb (1:100) for 24 h. PHA-preactivated primary T cells were restimulated with αCD3 (10 μg/ml) and TNF-α pAb for 24 h. Expression of FasL/Fas (A, B) and TRAIL/DR4/DR5 (C, D) was detected by flow cytometry. (E, F) Jurkat cells transfected with 100 nM siRNA for FasL or TRAIL for 48 h were activated with PHA-P (5 μg/ml) in the absence or presence of TNF-α pAb for 24 h. The mRNA levels of FasL and TRAIL were determined by Real-time qPCR (E) and the <t>apoptosis</t> was detected by Annexin V/PI (F). All the quantitative data represent means ± S.D. of at least three independent experiments. * p
    Apoptosis, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3046 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apoptosis/product/Cell Signaling Technology Inc
    Average 99 stars, based on 3046 article reviews
    Price from $9.99 to $1999.99
    apoptosis - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher mitochondrial membrane potential
    The reverse signaling of tmTNF-α promotes AICD through upregulating FasL/Fas and TRAIL/DR4 (A, B, C, D) Jurkat cells were simultaneously stimulated with PHA-P (5 μg/ml) and TNF-α pAb (1:100) for 24 h. PHA-preactivated primary T cells were restimulated with αCD3 (10 μg/ml) and TNF-α pAb for 24 h. Expression of FasL/Fas (A, B) and TRAIL/DR4/DR5 (C, D) was detected by flow cytometry. (E, F) Jurkat cells transfected with 100 nM siRNA for FasL or TRAIL for 48 h were activated with PHA-P (5 μg/ml) in the absence or presence of TNF-α pAb for 24 h. The mRNA levels of FasL and TRAIL were determined by Real-time qPCR (E) and the <t>apoptosis</t> was detected by Annexin V/PI (F). All the quantitative data represent means ± S.D. of at least three independent experiments. * p
    Mitochondrial Membrane Potential, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6414 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mitochondrial membrane potential/product/Thermo Fisher
    Average 99 stars, based on 6414 article reviews
    Price from $9.99 to $1999.99
    mitochondrial membrane potential - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    92
    Becton Dickinson apoptosis detection kit
    Cisplatin treatment in PKCδ silenced A375 human melanoma cells induces ceramide mediated <t>apoptosis</t> through IRF1-TNFα axis (A) 2 × 10 6 A375 cells were transfected with PKCδ specific siRNA or control siRNA as mentioned in Materials and Methods and the transfected cells were collected in TRIZOL for mRNA expression of PKCδ by semi quantitative RT-PCR. GAPDH was used as a reference. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P
    Apoptosis Detection Kit, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2528 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apoptosis detection kit/product/Becton Dickinson
    Average 92 stars, based on 2528 article reviews
    Price from $9.99 to $1999.99
    apoptosis detection kit - by Bioz Stars, 2020-11
    92/100 stars
      Buy from Supplier

    94
    Beyotime apoptosis
    Bcl-2 cannot block <t>apoptosis</t> induced by c-Bax. a Differential expression of Bax-HA and ΔBax-HA. AGS cells were transfected with 1 µg, 2 µg, or 4 µg of either Bax-HA or ΔBax-HA for 24 h and subjected to immunoblotting for protein expression. b Short-lived ΔBax-HA than Bax-HA. CHX (50 ug/ml) was added to 293 T or AGS cells transfected with plasmid encoding Bax-HA or ΔBax-HA prior to treatment and cultured for 24 h. Equivalent lysates from the cells harvested at the indicated time points were immunoblotted for indicated protein expression. c , d , g Bcl-2 did not decrease apoptosis induced by ΔBax-HA. 1 µg Bax-HA or 4 µg ΔBax-HA plasmid was transfected with 4 µg Bcl-2-Flag ( c ) or 4 µg ΔBax-HA and indicated quality of Bcl-2-Flag plasmid, then cultured for 24 h and AGS subjected to crystal violet staining or immunoblot ( c , g ). AGS cells were treated with 1 µg Bax-HA or 4 µg ΔBax-HA plasmid ( d ) and subjected to Annexin V/propidium iodide double staining and FCM to detect apoptosis. e , f Cleaved Bax was a stronger apoptosis inducer. e The tet-on system was constructed and protein expression was induced by 10uM Tetracyclines for 24 h in AGS. Apoptosis level and protein level was detected by FCM and immunoblot. f The AGS stable cell line was constructed and incubated with 50 nM MLN8237 for 72 h. Apoptosis level and protein level was detected by FCM and immunoblot. Error bars represent SD from three independent experiments. This immunoblot is representative of three independent experiments. Asterisk (*) indicates a significant difference. **** P
    Apoptosis, supplied by Beyotime, used in various techniques. Bioz Stars score: 94/100, based on 2262 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apoptosis/product/Beyotime
    Average 94 stars, based on 2262 article reviews
    Price from $9.99 to $1999.99
    apoptosis - by Bioz Stars, 2020-11
    94/100 stars
      Buy from Supplier

    99
    Thermo Fisher vybrant apoptosis assay kit
    Bcl-2 cannot block <t>apoptosis</t> induced by c-Bax. a Differential expression of Bax-HA and ΔBax-HA. AGS cells were transfected with 1 µg, 2 µg, or 4 µg of either Bax-HA or ΔBax-HA for 24 h and subjected to immunoblotting for protein expression. b Short-lived ΔBax-HA than Bax-HA. CHX (50 ug/ml) was added to 293 T or AGS cells transfected with plasmid encoding Bax-HA or ΔBax-HA prior to treatment and cultured for 24 h. Equivalent lysates from the cells harvested at the indicated time points were immunoblotted for indicated protein expression. c , d , g Bcl-2 did not decrease apoptosis induced by ΔBax-HA. 1 µg Bax-HA or 4 µg ΔBax-HA plasmid was transfected with 4 µg Bcl-2-Flag ( c ) or 4 µg ΔBax-HA and indicated quality of Bcl-2-Flag plasmid, then cultured for 24 h and AGS subjected to crystal violet staining or immunoblot ( c , g ). AGS cells were treated with 1 µg Bax-HA or 4 µg ΔBax-HA plasmid ( d ) and subjected to Annexin V/propidium iodide double staining and FCM to detect apoptosis. e , f Cleaved Bax was a stronger apoptosis inducer. e The tet-on system was constructed and protein expression was induced by 10uM Tetracyclines for 24 h in AGS. Apoptosis level and protein level was detected by FCM and immunoblot. f The AGS stable cell line was constructed and incubated with 50 nM MLN8237 for 72 h. Apoptosis level and protein level was detected by FCM and immunoblot. Error bars represent SD from three independent experiments. This immunoblot is representative of three independent experiments. Asterisk (*) indicates a significant difference. **** P
    Vybrant Apoptosis Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2338 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vybrant apoptosis assay kit/product/Thermo Fisher
    Average 99 stars, based on 2338 article reviews
    Price from $9.99 to $1999.99
    vybrant apoptosis assay kit - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    99
    Millipore apoptosis
    Acitretin preferentially induces <t>apoptosis</t> in HIV-infected cells. ( a ) Percentage of apoptotic cells determined by annexin V staining of GM-HIV-infected or mock-infected CD4+Tcells after 72 h of treatment. The percentage of apoptotic cells was significantly
    Apoptosis, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 17651 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apoptosis/product/Millipore
    Average 99 stars, based on 17651 article reviews
    Price from $9.99 to $1999.99
    apoptosis - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    94
    Becton Dickinson annexin v apoptosis detection kit
    Drp1 silencing attenuates CPT-induced mitochondrial fragmentation and <t>apoptosis.</t> a The effects of CPT on mitochondrial fusion/fission proteins expression in OS cells. Representative immunoblot of the protein levels of Drp1, Opa1, Mfn1 and Mfn2. b Comparison of mitochondrial morphology in the control and Drp1 siRNA cells. Images of mitochondria (red) and nucleus (blue) were collected by confocal microscope. c Drp1 silencing reversed the reduction of cell viability in CPT treated 143B cells. Representative dot plots of Annexin V/PI analysis after CPT treatments in the presence or absence of Drp1 siRNA. d CPT decreased ATP production and increased ADP/ATP ratio of both 143B and MG63 cells. The results were expressed as the means ± SD from three independent experiments. * P
    Annexin V Apoptosis Detection Kit, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 2103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/annexin v apoptosis detection kit/product/Becton Dickinson
    Average 94 stars, based on 2103 article reviews
    Price from $9.99 to $1999.99
    annexin v apoptosis detection kit - by Bioz Stars, 2020-11
    94/100 stars
      Buy from Supplier

    99
    Millipore annexin v fitc apoptosis detection kit
    Drp1 silencing attenuates CPT-induced mitochondrial fragmentation and <t>apoptosis.</t> a The effects of CPT on mitochondrial fusion/fission proteins expression in OS cells. Representative immunoblot of the protein levels of Drp1, Opa1, Mfn1 and Mfn2. b Comparison of mitochondrial morphology in the control and Drp1 siRNA cells. Images of mitochondria (red) and nucleus (blue) were collected by confocal microscope. c Drp1 silencing reversed the reduction of cell viability in CPT treated 143B cells. Representative dot plots of Annexin V/PI analysis after CPT treatments in the presence or absence of Drp1 siRNA. d CPT decreased ATP production and increased ADP/ATP ratio of both 143B and MG63 cells. The results were expressed as the means ± SD from three independent experiments. * P
    Annexin V Fitc Apoptosis Detection Kit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/annexin v fitc apoptosis detection kit/product/Millipore
    Average 99 stars, based on 3130 article reviews
    Price from $9.99 to $1999.99
    annexin v fitc apoptosis detection kit - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    99
    BioVision apoptosis
    Effect of miR-200b-3p on the proliferation and <t>apoptosis</t> of CRC cells. (A) CFSE assay for cell proliferation. (B) Flow cytometric analysis for apoptosis. **P
    Apoptosis, supplied by BioVision, used in various techniques. Bioz Stars score: 99/100, based on 2037 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apoptosis/product/BioVision
    Average 99 stars, based on 2037 article reviews
    Price from $9.99 to $1999.99
    apoptosis - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    93
    Abcam apoptosis
    p53 expression affects the <t>apoptosis</t> rate of hemangioma-derived endothelial cells treated with propranolol (100 µmol/l). *P
    Apoptosis, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 1595 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apoptosis/product/Abcam
    Average 93 stars, based on 1595 article reviews
    Price from $9.99 to $1999.99
    apoptosis - by Bioz Stars, 2020-11
    93/100 stars
      Buy from Supplier

    94
    Keygen Biotech apoptosis
    Characterization of apoptotic signaling efficiency of truncated variants of Gal-3 (A) Schematic details of Gal-3 and its variants. (B) SDS-PAGE analysis of Gal-3 and its variants. (C-D) Jurkat cells were treated with 2.5 μM Gal-3 or its variants for 18 h and (C) cellular <t>apoptosis</t> was assessed by flow cytometry and (D) signaling was assessed by western blot analysis of p-ERK1/2 and cleaved caspase-3.
    Apoptosis, supplied by Keygen Biotech, used in various techniques. Bioz Stars score: 94/100, based on 1990 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apoptosis/product/Keygen Biotech
    Average 94 stars, based on 1990 article reviews
    Price from $9.99 to $1999.99
    apoptosis - by Bioz Stars, 2020-11
    94/100 stars
      Buy from Supplier

    99
    TaKaRa in situ apoptosis detection kit
    Characterization of apoptotic signaling efficiency of truncated variants of Gal-3 (A) Schematic details of Gal-3 and its variants. (B) SDS-PAGE analysis of Gal-3 and its variants. (C-D) Jurkat cells were treated with 2.5 μM Gal-3 or its variants for 18 h and (C) cellular <t>apoptosis</t> was assessed by flow cytometry and (D) signaling was assessed by western blot analysis of p-ERK1/2 and cleaved caspase-3.
    In Situ Apoptosis Detection Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1831 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/in situ apoptosis detection kit/product/TaKaRa
    Average 99 stars, based on 1831 article reviews
    Price from $9.99 to $1999.99
    in situ apoptosis detection kit - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    Image Search Results


    Concomitant loss of MLKL and Caspase-8, but not loss of RIPK1 kinase activity or combined loss of RIPK3 and Caspase-8, promotes survival of LUBAC-deficient mice a, Representative images of E10.5 ( n =6 embryos/genotype), scale bar: 2 mm, E14.5 ( n =12 Ripk1 K45A Hoil-1 +/- , n =5 Ripk1 K45A Hoil-1 -/- embryos/genotype) and E15.5 embryos ( n =3 embryos/genotype). Scale bar: 5 mm. *: poor yolk sac vascularisation. b, f, Representative images of yolk sac vascularisation (PECAM-1, red) and apoptosis (cleaved (cl.) Caspase-3, green) at E14.5 (b) or E13.5 (f) and quantification. Mean ± s.e.m. and P values from unpaired two-tailed t -tests (b) or one-way ANOVA (f) are shown. Scale bar: 50 µm. c, FADD-IP in MEFs treated for 3 h with zVAD-fmk ± TNF ( n . d, Cell death by PI incorporation in MEFs ± TNF (10 ng/ml) or LT-α. Mean ± s.e.m. ( n =3 independent experiments) and P values (**** P

    Journal: Nature

    Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

    doi: 10.1038/s41586-018-0064-8

    Figure Lengend Snippet: Concomitant loss of MLKL and Caspase-8, but not loss of RIPK1 kinase activity or combined loss of RIPK3 and Caspase-8, promotes survival of LUBAC-deficient mice a, Representative images of E10.5 ( n =6 embryos/genotype), scale bar: 2 mm, E14.5 ( n =12 Ripk1 K45A Hoil-1 +/- , n =5 Ripk1 K45A Hoil-1 -/- embryos/genotype) and E15.5 embryos ( n =3 embryos/genotype). Scale bar: 5 mm. *: poor yolk sac vascularisation. b, f, Representative images of yolk sac vascularisation (PECAM-1, red) and apoptosis (cleaved (cl.) Caspase-3, green) at E14.5 (b) or E13.5 (f) and quantification. Mean ± s.e.m. and P values from unpaired two-tailed t -tests (b) or one-way ANOVA (f) are shown. Scale bar: 50 µm. c, FADD-IP in MEFs treated for 3 h with zVAD-fmk ± TNF ( n . d, Cell death by PI incorporation in MEFs ± TNF (10 ng/ml) or LT-α. Mean ± s.e.m. ( n =3 independent experiments) and P values (**** P

    Article Snippet: For whole mount TUNEL staining and immunofluorescence staining, samples were processed using the ApoTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7101) according to the manufacturer’s instructions and as previously described .

    Techniques: Activity Assay, Mouse Assay, Two Tailed Test

    TNFR1 signalling drives cell death and lethality of HOIL-1-deficient mice at mid-gestation. a, d , Quantification of genotypes of animals obtained from inter-crosses of Tnf -/- Hoil-1 +/- (a) and Tnfr1 -/- Hoil-1 +/- (d) mice. *: dead embryos. b, Representative images of embryos quantified in (a) at E10.5 and E15.5, *; poor yolk sac. c, Cell death as detected by whole-mount TUNEL staining in yolk sacs at E10.5 (n= 3 embryos/genotype). e, Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved Caspase-3, bottom panel) of yolk sacs. Merged image is shown in Scale bar: 50 µm. f, Representative images of embryos at E16.5 ( n =2 for Tnfr1 -/- Hoil-1 +/- and n =4 for Tnfr1 -/- Hoil-1 -/- ). Fig. 1g.

    Journal: Nature

    Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

    doi: 10.1038/s41586-018-0064-8

    Figure Lengend Snippet: TNFR1 signalling drives cell death and lethality of HOIL-1-deficient mice at mid-gestation. a, d , Quantification of genotypes of animals obtained from inter-crosses of Tnf -/- Hoil-1 +/- (a) and Tnfr1 -/- Hoil-1 +/- (d) mice. *: dead embryos. b, Representative images of embryos quantified in (a) at E10.5 and E15.5, *; poor yolk sac. c, Cell death as detected by whole-mount TUNEL staining in yolk sacs at E10.5 (n= 3 embryos/genotype). e, Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved Caspase-3, bottom panel) of yolk sacs. Merged image is shown in Scale bar: 50 µm. f, Representative images of embryos at E16.5 ( n =2 for Tnfr1 -/- Hoil-1 +/- and n =4 for Tnfr1 -/- Hoil-1 -/- ). Fig. 1g.

    Article Snippet: For whole mount TUNEL staining and immunofluorescence staining, samples were processed using the ApoTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7101) according to the manufacturer’s instructions and as previously described .

    Techniques: Mouse Assay, TUNEL Assay, Staining

    HOIL-1-deficient mice die at mid-gestation a, Schematic representation of the Hoil-1 knockout strategy. Solid boxes represent Hoil-1 exons and grey boxes with a star indicate the targeted exons. Boxes with diagonal and horizontal strips represent LoxP and Frt sites, respectively. b, Specificity of gene recombination was assessed by Southern blotting with 5’ and 3’ probes external to the construct in four clones (14B8, 14F6, 20D7 and 21F7). Digest of the DNA with ApaI, followed by hybridisation with the 3’ probe was expected to show a 5700 bp band for the WT allele and a 7700 bp band for the mutant allele. All four clones appeared to have the correct recombination on the 3’ side. Digest of the DNA with SphI and hybridisation with the 5’ probe was expected to show a 4500 bp WT band and a 6200 bp band for the mutated allele. Clones 14B8, 14F6 and 21F7 appeared to be correctly recombined on the 5’ side. Finally, cutting the DNA with ApaI and hybridising with a hygro probe showed a single band in all clones, indicative of a single integration of the construct in all four ES clones. Clones 14B8 and 14F6 were selected for generation of the two Hoil-1 -/- strains. c , PCR analysis of Hoil-1 wild-type, heterozygous and knockout mice. d , Protein levels of HOIL-1, HOIP and SHARPIN in whole embryo lysates ( n = 3 for Hoil-1 +/- and Hoil-1 -/- embryos and n =1 for Hoil-1 +/+ . e , Quantification of genotypes of animals obtained from inter-crossing C20Hoil-1 +/- mice. *indicates dead embryos. f , Representative images of C20Hoil-1 +/- and C20Hoil-1 -/- embryos from E9.5 to E11.5 as quantified in (e). Scale bar: 2 mm. g, Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved Caspase-3, bottom panel) of yolk sacs. Merged image is shown in . h , Whole-mount TUNEL staining of embryos at the indicated stages (embryo/genotype n =2 at E10.5, n =8 for Hoil-1 +/- and n =5 for Hoil-1 -/- at E11.5). Scale bar: 2 mm. i, Quantification of genotypes of animals obtained from inter-crossing Hoil-1 fl/wt Tie2-Cre + with Hoil-1 fl/fl Tie2-Cre - mice. *indicates dead embryos. j , Representative images of embryos with conditional deletion of Hoil-1 in Tie2-Cre expressing cells as quantified in (i). Scale bar: 2 mm. *: poorly vascularised yolk sac. Fig. 1c

    Journal: Nature

    Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

    doi: 10.1038/s41586-018-0064-8

    Figure Lengend Snippet: HOIL-1-deficient mice die at mid-gestation a, Schematic representation of the Hoil-1 knockout strategy. Solid boxes represent Hoil-1 exons and grey boxes with a star indicate the targeted exons. Boxes with diagonal and horizontal strips represent LoxP and Frt sites, respectively. b, Specificity of gene recombination was assessed by Southern blotting with 5’ and 3’ probes external to the construct in four clones (14B8, 14F6, 20D7 and 21F7). Digest of the DNA with ApaI, followed by hybridisation with the 3’ probe was expected to show a 5700 bp band for the WT allele and a 7700 bp band for the mutant allele. All four clones appeared to have the correct recombination on the 3’ side. Digest of the DNA with SphI and hybridisation with the 5’ probe was expected to show a 4500 bp WT band and a 6200 bp band for the mutated allele. Clones 14B8, 14F6 and 21F7 appeared to be correctly recombined on the 5’ side. Finally, cutting the DNA with ApaI and hybridising with a hygro probe showed a single band in all clones, indicative of a single integration of the construct in all four ES clones. Clones 14B8 and 14F6 were selected for generation of the two Hoil-1 -/- strains. c , PCR analysis of Hoil-1 wild-type, heterozygous and knockout mice. d , Protein levels of HOIL-1, HOIP and SHARPIN in whole embryo lysates ( n = 3 for Hoil-1 +/- and Hoil-1 -/- embryos and n =1 for Hoil-1 +/+ . e , Quantification of genotypes of animals obtained from inter-crossing C20Hoil-1 +/- mice. *indicates dead embryos. f , Representative images of C20Hoil-1 +/- and C20Hoil-1 -/- embryos from E9.5 to E11.5 as quantified in (e). Scale bar: 2 mm. g, Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved Caspase-3, bottom panel) of yolk sacs. Merged image is shown in . h , Whole-mount TUNEL staining of embryos at the indicated stages (embryo/genotype n =2 at E10.5, n =8 for Hoil-1 +/- and n =5 for Hoil-1 -/- at E11.5). Scale bar: 2 mm. i, Quantification of genotypes of animals obtained from inter-crossing Hoil-1 fl/wt Tie2-Cre + with Hoil-1 fl/fl Tie2-Cre - mice. *indicates dead embryos. j , Representative images of embryos with conditional deletion of Hoil-1 in Tie2-Cre expressing cells as quantified in (i). Scale bar: 2 mm. *: poorly vascularised yolk sac. Fig. 1c

    Article Snippet: For whole mount TUNEL staining and immunofluorescence staining, samples were processed using the ApoTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7101) according to the manufacturer’s instructions and as previously described .

    Techniques: Mouse Assay, Knock-Out, Southern Blot, Construct, Clone Assay, Hybridization, Mutagenesis, Polymerase Chain Reaction, Staining, TUNEL Assay, Expressing

    Individual deletion of mediators of apoptosis or necroptosis does not prevent cell death and lethality at mid-gestation of HOIL-1- or HOIP-deficient embryos. a , Western blot analysis of MLKL expression in the indicated organs derived from control Mlkl -/- mice ( n . b, d, e, f, Representative images of embryos at different stages of gestation (E10.5: n =7 for Ripk3 -/- Hoil-1 +/- and n =5 for Ripk3 -/- Hoil-1 -/- ; E11.5: n =5 for Ripk3 -/- Hoil-1 +/- and n =2 for Ripk3 -/- Hoil-1 -/- ; E12.5: n =9 for Ripk3 -/- Hoil-1 +/- and n =2 for Ripk3 -/- Hoil-1 -/- (b), E10.5: n =16 for Mlkl -/- Hoip +/- and n =6 for Mlkl -/- Hoip -/- ; E11.5: n =8 for Mlkl -/- Hoip +/- and n =6 for Mlkl -/- Hoip -/- ; E12.5: n =10 for Mlkl -/- Hoip +/- and n =5 for Mlkl -/- Hoip -/- (d), E10.5: n =5 for Caspase-8 +/- Hoip +/- and n =4 for Caspase-8 +/- Hoip -/- ; E11.5: n =6 for Caspase-8 +/- Hoip +/- and n =3 for Caspase-8 +/- Hoip -/- ; E12.5: n =3 for Caspase-8 +/- Hoip +/- and n =2 for Caspase-8 +/- Hoip -/- (e), E10.5: n =2 for Caspase-8 +/- Hoil-1 +/- and n =4 for Caspase-8 +/- Hoil-1 -/- ; E11.5: n =2 for Caspase-8 +/- Hoil-1 +/- and n =5 for Caspase-8 +/- Hoil-1 -/- ; E12.5: n =6 for Caspase-8 +/- Hoil-1 +/- and n =3 for Caspase-8 +/- Hoil-1 -/- (f)). *: poor yolk sac vascularisation. Scale bar: 2 mm. c, Representative images of yolk sac vascularisation and cell death at E10.5 as detected by PECAM-1 (red) and cleaved (cl.) Caspase-3 staining (green) (top panel) and whole mount TUNEL staining (bottom panel) ( n =4 per genotype). Scale bar: 50 µm.

    Journal: Nature

    Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

    doi: 10.1038/s41586-018-0064-8

    Figure Lengend Snippet: Individual deletion of mediators of apoptosis or necroptosis does not prevent cell death and lethality at mid-gestation of HOIL-1- or HOIP-deficient embryos. a , Western blot analysis of MLKL expression in the indicated organs derived from control Mlkl -/- mice ( n . b, d, e, f, Representative images of embryos at different stages of gestation (E10.5: n =7 for Ripk3 -/- Hoil-1 +/- and n =5 for Ripk3 -/- Hoil-1 -/- ; E11.5: n =5 for Ripk3 -/- Hoil-1 +/- and n =2 for Ripk3 -/- Hoil-1 -/- ; E12.5: n =9 for Ripk3 -/- Hoil-1 +/- and n =2 for Ripk3 -/- Hoil-1 -/- (b), E10.5: n =16 for Mlkl -/- Hoip +/- and n =6 for Mlkl -/- Hoip -/- ; E11.5: n =8 for Mlkl -/- Hoip +/- and n =6 for Mlkl -/- Hoip -/- ; E12.5: n =10 for Mlkl -/- Hoip +/- and n =5 for Mlkl -/- Hoip -/- (d), E10.5: n =5 for Caspase-8 +/- Hoip +/- and n =4 for Caspase-8 +/- Hoip -/- ; E11.5: n =6 for Caspase-8 +/- Hoip +/- and n =3 for Caspase-8 +/- Hoip -/- ; E12.5: n =3 for Caspase-8 +/- Hoip +/- and n =2 for Caspase-8 +/- Hoip -/- (e), E10.5: n =2 for Caspase-8 +/- Hoil-1 +/- and n =4 for Caspase-8 +/- Hoil-1 -/- ; E11.5: n =2 for Caspase-8 +/- Hoil-1 +/- and n =5 for Caspase-8 +/- Hoil-1 -/- ; E12.5: n =6 for Caspase-8 +/- Hoil-1 +/- and n =3 for Caspase-8 +/- Hoil-1 -/- (f)). *: poor yolk sac vascularisation. Scale bar: 2 mm. c, Representative images of yolk sac vascularisation and cell death at E10.5 as detected by PECAM-1 (red) and cleaved (cl.) Caspase-3 staining (green) (top panel) and whole mount TUNEL staining (bottom panel) ( n =4 per genotype). Scale bar: 50 µm.

    Article Snippet: For whole mount TUNEL staining and immunofluorescence staining, samples were processed using the ApoTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7101) according to the manufacturer’s instructions and as previously described .

    Techniques: Western Blot, Expressing, Derivative Assay, Mouse Assay, Staining, TUNEL Assay

    Combined deletion of MLKL and Caspase-8 promotes survival of LUBAC-deficient mice. a, Quantification of genotypes of animals obtained from inter-crosses of Mlkl -/- Caspase-8 +/- Hoip +/- with Mlkl -/- Caspase-8 -/- Hoip +/- mice. *: dead embryos. b, Representative images of adult mice as quantified in (a). c , Kaplan-Meier plot of mouse survival ( n =6 for Mlkl -/- Caspase-8 -/- Hoip -/- and n =9 for Mlkl -/- Caspase-8 -/- Hoil-1 -/- mice). d, Representative images of H E staining of the indicated organs ( n =3 mice/genotype). Scale bar: 200 µm. e, Representative images of yolk sac vascularisation (PECAM-1, red) and apoptosis (cleaved (cl.) Caspase-3, green) (top panel) at E13.5 and respective quantifications (bottom panel). Mean ± s.e.m ( n =5 for Mlkl -/- Caspase-8 -/- Hoil-1 +/- and Mlkl -/- Caspase-8 -/- Hoil-1 -/- and n =2 for Mlkl -/- Caspase-8 +/- Hoil-1 -/- ) are shown and results were analysed with unpaired two-tailed t -tests comparing Mlkl -/- Caspase-8 -/- Hoil-1 +/- and Mlkl -/- Caspase-8 -/- Hoil-1 -/- embryos. f, Representative images of H E staining of the indicated organs ( n =3 embryos/genotype). Scale bar: 200 µm. g, Epidermal thickness quantification of mice of the indicated genotypes in (f). Mean ± s.e.m values ( n =3 mice/genotype) are shown and results were analysed with unpaired two-tailed t -tests. h, Western blot analysis of lysates from whole E13.5 embryos of the indicated genotypes as well as L929 cells treated or not with TNF plus zVAD-fmk for 2 h as antibody validation ( n .

    Journal: Nature

    Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

    doi: 10.1038/s41586-018-0064-8

    Figure Lengend Snippet: Combined deletion of MLKL and Caspase-8 promotes survival of LUBAC-deficient mice. a, Quantification of genotypes of animals obtained from inter-crosses of Mlkl -/- Caspase-8 +/- Hoip +/- with Mlkl -/- Caspase-8 -/- Hoip +/- mice. *: dead embryos. b, Representative images of adult mice as quantified in (a). c , Kaplan-Meier plot of mouse survival ( n =6 for Mlkl -/- Caspase-8 -/- Hoip -/- and n =9 for Mlkl -/- Caspase-8 -/- Hoil-1 -/- mice). d, Representative images of H E staining of the indicated organs ( n =3 mice/genotype). Scale bar: 200 µm. e, Representative images of yolk sac vascularisation (PECAM-1, red) and apoptosis (cleaved (cl.) Caspase-3, green) (top panel) at E13.5 and respective quantifications (bottom panel). Mean ± s.e.m ( n =5 for Mlkl -/- Caspase-8 -/- Hoil-1 +/- and Mlkl -/- Caspase-8 -/- Hoil-1 -/- and n =2 for Mlkl -/- Caspase-8 +/- Hoil-1 -/- ) are shown and results were analysed with unpaired two-tailed t -tests comparing Mlkl -/- Caspase-8 -/- Hoil-1 +/- and Mlkl -/- Caspase-8 -/- Hoil-1 -/- embryos. f, Representative images of H E staining of the indicated organs ( n =3 embryos/genotype). Scale bar: 200 µm. g, Epidermal thickness quantification of mice of the indicated genotypes in (f). Mean ± s.e.m values ( n =3 mice/genotype) are shown and results were analysed with unpaired two-tailed t -tests. h, Western blot analysis of lysates from whole E13.5 embryos of the indicated genotypes as well as L929 cells treated or not with TNF plus zVAD-fmk for 2 h as antibody validation ( n .

    Article Snippet: For whole mount TUNEL staining and immunofluorescence staining, samples were processed using the ApoTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7101) according to the manufacturer’s instructions and as previously described .

    Techniques: Mouse Assay, Staining, Two Tailed Test, Western Blot

    Combined deletion of RIPK3 and Caspase-8 prevents cell death but not embryonic lethality at late gestation that is caused by the loss of HOIL-1. a , Quantification of genotypes of animals obtained from inter-crosses of Ripk3 -/- Caspase-8 +/- Hoil-1 +/- with Ripk3 -/- Caspase-8 -/- Hoil-1 +/- mice (left panel) or Ripk3 -/- Caspase-8 -/- Hoil-1 +/- mice (right panel). b, Health status of Ripk3 -/- Caspase-8 +/- Hoil-1 -/- and Ripk3 -/- Caspase-8 -/- Hoil-1 -/- embryos at different developmental stages. c , Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved (cl.) Caspase-3, bottom panel) of yolk sacs. Merged image is shown in . Scale bar: 50 µm. d, Cell death as detected by whole-mount TUNEL staining in yolk sacs at E14.5 (left panel) and respective quantification (right panel). Mean ± s.e.m. ( n =3 embryos/genotype) and P values from one-way ANOVA are reported. e, f Representative images (e) and quantification (f) of cell death in different organs as detected by TUNEL staining at E13.5 ( n =3 embryos/genotype) and E14.5 ( n =5 for Ripk3 -/- Caspase-8 -/- Hoil-1 +/- , n=2 for Ripk3 -/- Caspase-8 -/- Hoil-1 -/- and Ripk3 -/- Caspase-8 -/- Hoil-1 -/- lung and liver and n =3 Ripk3 -/- Caspase-8 -/- Hoil-1 -/- heart). Scale bar: 50 µm (e). Mean ± s.e.m. values are shown (f). g, Cell death was analysed by PI staining in MEFs stimulated or not with the indicated ligands for 24 h. Mean ± s.e.m. ( n =3 independent experiments) and P values from two-way ANOVA are reported. h, Representative images of H E staining on E13.5 whole-embryo paraffin embedded sections ( n =3 for Ripk3 -/- Caspase-8 -/- Hoil-1 +/- and Ripk3 -/- Caspase-8 -/- Hoil-1 -/- and n =2 for Ripk3 -/- Caspase-8 +/- Hoil-1 -/- ). *: pericardial effusion, arrows; congested vessels H, heart; L, lung; Li, liver. Scale bar: 200 µm. i, Representative images of micro-focus CT scan images of whole E13.5 embryos ( n =3 embryos/genotype). *: pericardial effusion Fig. 3f

    Journal: Nature

    Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

    doi: 10.1038/s41586-018-0064-8

    Figure Lengend Snippet: Combined deletion of RIPK3 and Caspase-8 prevents cell death but not embryonic lethality at late gestation that is caused by the loss of HOIL-1. a , Quantification of genotypes of animals obtained from inter-crosses of Ripk3 -/- Caspase-8 +/- Hoil-1 +/- with Ripk3 -/- Caspase-8 -/- Hoil-1 +/- mice (left panel) or Ripk3 -/- Caspase-8 -/- Hoil-1 +/- mice (right panel). b, Health status of Ripk3 -/- Caspase-8 +/- Hoil-1 -/- and Ripk3 -/- Caspase-8 -/- Hoil-1 -/- embryos at different developmental stages. c , Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved (cl.) Caspase-3, bottom panel) of yolk sacs. Merged image is shown in . Scale bar: 50 µm. d, Cell death as detected by whole-mount TUNEL staining in yolk sacs at E14.5 (left panel) and respective quantification (right panel). Mean ± s.e.m. ( n =3 embryos/genotype) and P values from one-way ANOVA are reported. e, f Representative images (e) and quantification (f) of cell death in different organs as detected by TUNEL staining at E13.5 ( n =3 embryos/genotype) and E14.5 ( n =5 for Ripk3 -/- Caspase-8 -/- Hoil-1 +/- , n=2 for Ripk3 -/- Caspase-8 -/- Hoil-1 -/- and Ripk3 -/- Caspase-8 -/- Hoil-1 -/- lung and liver and n =3 Ripk3 -/- Caspase-8 -/- Hoil-1 -/- heart). Scale bar: 50 µm (e). Mean ± s.e.m. values are shown (f). g, Cell death was analysed by PI staining in MEFs stimulated or not with the indicated ligands for 24 h. Mean ± s.e.m. ( n =3 independent experiments) and P values from two-way ANOVA are reported. h, Representative images of H E staining on E13.5 whole-embryo paraffin embedded sections ( n =3 for Ripk3 -/- Caspase-8 -/- Hoil-1 +/- and Ripk3 -/- Caspase-8 -/- Hoil-1 -/- and n =2 for Ripk3 -/- Caspase-8 +/- Hoil-1 -/- ). *: pericardial effusion, arrows; congested vessels H, heart; L, lung; Li, liver. Scale bar: 200 µm. i, Representative images of micro-focus CT scan images of whole E13.5 embryos ( n =3 embryos/genotype). *: pericardial effusion Fig. 3f

    Article Snippet: For whole mount TUNEL staining and immunofluorescence staining, samples were processed using the ApoTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7101) according to the manufacturer’s instructions and as previously described .

    Techniques: Mouse Assay, Staining, TUNEL Assay, Computed Tomography

    Ablation of the kinase activity of RIPK1 in HOIL-1- or HOIP-deficient embryos prevents cell death and lethality at mid-gestation but not at late gestation. a, b, Quantification of genotypes of animals obtained after inter-crossing Ripk1 K45A Hoil-1 +/- (a) and Ripk1 K45A Hoip +/- (b) mice. *indicates dead embryos. c, Representative images of embryos quantified in (b) *; poor yolk sac vascularisation. Scale bar: 2 mm. d, Whole-mount TUNEL staining of embryos ( n =2 embryos). Scale bar: 2 mm. e, Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved (cl.) Caspase-3, bottom panel) of yolk sacs. Merged image is shown in . f, g, Representative images of cell death in different organs (f) and quantification (g) as detected by TUNEL staining at E14.5 ( n =3 embryos/genotype). Scale bar: 50 μm (f). Mean ± s.e.m. ( n =3 embryos/genotype) and P values from one-way ANOVA are reported (g). h, Representative images of H E staining on whole-embryo paraffin sections ( n =3 embryos/genotype). *; pericardial effusion, n, necrotic area. H, heart; L, lung; Li, liver. Scale bar: 200 µm. i, Cell death was analysed by PI staining in MEFs stimulated or not with TNF for 24 h plus the indicated cell death inhibitors. Mean ± s.e.m. ( n =3 independent experiments) and P values from two-way ANOVA are reported. Fig. 3b

    Journal: Nature

    Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

    doi: 10.1038/s41586-018-0064-8

    Figure Lengend Snippet: Ablation of the kinase activity of RIPK1 in HOIL-1- or HOIP-deficient embryos prevents cell death and lethality at mid-gestation but not at late gestation. a, b, Quantification of genotypes of animals obtained after inter-crossing Ripk1 K45A Hoil-1 +/- (a) and Ripk1 K45A Hoip +/- (b) mice. *indicates dead embryos. c, Representative images of embryos quantified in (b) *; poor yolk sac vascularisation. Scale bar: 2 mm. d, Whole-mount TUNEL staining of embryos ( n =2 embryos). Scale bar: 2 mm. e, Single staining showing vascularisation (PECAM-1, top panel) and apoptosis (cleaved (cl.) Caspase-3, bottom panel) of yolk sacs. Merged image is shown in . f, g, Representative images of cell death in different organs (f) and quantification (g) as detected by TUNEL staining at E14.5 ( n =3 embryos/genotype). Scale bar: 50 μm (f). Mean ± s.e.m. ( n =3 embryos/genotype) and P values from one-way ANOVA are reported (g). h, Representative images of H E staining on whole-embryo paraffin sections ( n =3 embryos/genotype). *; pericardial effusion, n, necrotic area. H, heart; L, lung; Li, liver. Scale bar: 200 µm. i, Cell death was analysed by PI staining in MEFs stimulated or not with TNF for 24 h plus the indicated cell death inhibitors. Mean ± s.e.m. ( n =3 independent experiments) and P values from two-way ANOVA are reported. Fig. 3b

    Article Snippet: For whole mount TUNEL staining and immunofluorescence staining, samples were processed using the ApoTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7101) according to the manufacturer’s instructions and as previously described .

    Techniques: Activity Assay, Mouse Assay, TUNEL Assay, Staining

    HOIL-1 deficiency causes embryonic lethality at mid-gestation due to TNFR1-mediated endothelial cell death a, Mendelian frequencies obtained from inter-crossing Hoil-1 +/- mice, *: dead embryos. b, Representative images of embryos from E9.5 to E11.5 quantified in (a), *: poor yolk sac vascularisation. Scale bar: 2 mm. c, Representative images of yolk sac vascularisation (PECAM-1, red) and cell death (cleaved (cl.) Caspase-3 staining, green) at E10.5 (top panel) ( n =4 yolk-sacs/genotype), and whole-mount TUNEL staining (bottom, panel) ( n =2 yolk-sacs/genotype). Scale bar: 50 µm. d, g, Quantification of branching points (g) and cleaved Caspase-3 positive cells (g). Mean ± s.e.m. values and P values from unpaired two-tailed t -tests are shown. e, Representative images of embryos at E10.5 ( n =14 Hoil-1 fl/wt Tie2-Cre+ and n =7 Hoil-1 fl/fl Tie2-Cre+ embryos, top panel). *: poor yolk sac vascularisation. Scale bar: 2 mm. Yolk sac vascularisation (PECAM-1, red) and apoptosis (cleaved Caspase-3, green) (middle panel). Scale bar: 50 µm. Yolk sac whole-mount TUNEL staining ( n =6 Hoil-1 fl/wt Tie2-Cre+ and n =2 Hoil-1 fl/fl Tie2-Cre+ yolk-sacs/genotype , bottom panel). f , Representative images of embryos at E15.5 (top panel, n =6 Tnfr1 -/- Hoil-1 -/- and n =19 Tnfr1 -/- Hoil-1 +/- embryos), scale bar: 2 mm, and yolk sac vascularisation (PECAM-1, red) and apoptosis (cleaved Caspase-3, green) (bottom panel), Scale bar: 50 µm. h, Representative images of H E staining on whole-embryo paraffin sections ( n =3 embryos/genotype). *: pericardial effusion, arrows; congested vessels. H, heart; L, lung; Li, liver. Scale bar: 50 µm.

    Journal: Nature

    Article Title: LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis

    doi: 10.1038/s41586-018-0064-8

    Figure Lengend Snippet: HOIL-1 deficiency causes embryonic lethality at mid-gestation due to TNFR1-mediated endothelial cell death a, Mendelian frequencies obtained from inter-crossing Hoil-1 +/- mice, *: dead embryos. b, Representative images of embryos from E9.5 to E11.5 quantified in (a), *: poor yolk sac vascularisation. Scale bar: 2 mm. c, Representative images of yolk sac vascularisation (PECAM-1, red) and cell death (cleaved (cl.) Caspase-3 staining, green) at E10.5 (top panel) ( n =4 yolk-sacs/genotype), and whole-mount TUNEL staining (bottom, panel) ( n =2 yolk-sacs/genotype). Scale bar: 50 µm. d, g, Quantification of branching points (g) and cleaved Caspase-3 positive cells (g). Mean ± s.e.m. values and P values from unpaired two-tailed t -tests are shown. e, Representative images of embryos at E10.5 ( n =14 Hoil-1 fl/wt Tie2-Cre+ and n =7 Hoil-1 fl/fl Tie2-Cre+ embryos, top panel). *: poor yolk sac vascularisation. Scale bar: 2 mm. Yolk sac vascularisation (PECAM-1, red) and apoptosis (cleaved Caspase-3, green) (middle panel). Scale bar: 50 µm. Yolk sac whole-mount TUNEL staining ( n =6 Hoil-1 fl/wt Tie2-Cre+ and n =2 Hoil-1 fl/fl Tie2-Cre+ yolk-sacs/genotype , bottom panel). f , Representative images of embryos at E15.5 (top panel, n =6 Tnfr1 -/- Hoil-1 -/- and n =19 Tnfr1 -/- Hoil-1 +/- embryos), scale bar: 2 mm, and yolk sac vascularisation (PECAM-1, red) and apoptosis (cleaved Caspase-3, green) (bottom panel), Scale bar: 50 µm. h, Representative images of H E staining on whole-embryo paraffin sections ( n =3 embryos/genotype). *: pericardial effusion, arrows; congested vessels. H, heart; L, lung; Li, liver. Scale bar: 50 µm.

    Article Snippet: For whole mount TUNEL staining and immunofluorescence staining, samples were processed using the ApoTag plus Peroxidase In Situ Apoptosis Detection Kit (Millipore, S7101) according to the manufacturer’s instructions and as previously described .

    Techniques: Mouse Assay, Staining, TUNEL Assay, Two Tailed Test

    MPA but not NET-A or P4 increases Vpr-mediated apoptosis in a dose-dependent manner. (A) PBMCs were treated with or without 5 µM Vpr peptide (amino acids 52–96) and increasing concentrations of MPA, NET-A or P4 for 24 hrs. The graph shows results pooled from two independent experiments with samples from three donors. (B) PBMCs were treated with 100 nM MPA, 10 µM NET-A, 1 µM P4 or in combination with 5 µM Vpr peptide (amino acids 52–96) for 24 hrs. Cells were stained and acquired by flow cytometry as described in the materials and methods . A tryptic BSA digest served as a control wherever Vpr peptide was not added, as for results in figure 4 . The histogram shows results pooled from two independent experiments with samples from three different donors compared to those in figure A. donors. Statistical trend analysis for panel A was performed by the Wilcox rank-sum test, as further extended by Cuzick [85] , and showed a significant trend only for MPA plus Vpr (p = 0.012) and P4 minus Vpr (p = 0.012). Statistical significance in panel B was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: MPA but not NET-A or P4 increases Vpr-mediated apoptosis in a dose-dependent manner. (A) PBMCs were treated with or without 5 µM Vpr peptide (amino acids 52–96) and increasing concentrations of MPA, NET-A or P4 for 24 hrs. The graph shows results pooled from two independent experiments with samples from three donors. (B) PBMCs were treated with 100 nM MPA, 10 µM NET-A, 1 µM P4 or in combination with 5 µM Vpr peptide (amino acids 52–96) for 24 hrs. Cells were stained and acquired by flow cytometry as described in the materials and methods . A tryptic BSA digest served as a control wherever Vpr peptide was not added, as for results in figure 4 . The histogram shows results pooled from two independent experiments with samples from three different donors compared to those in figure A. donors. Statistical trend analysis for panel A was performed by the Wilcox rank-sum test, as further extended by Cuzick [85] , and showed a significant trend only for MPA plus Vpr (p = 0.012) and P4 minus Vpr (p = 0.012). Statistical significance in panel B was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Staining, Flow Cytometry, Cytometry

    HIV-1-mediated apoptosis is enhanced in the presence of Dex and MPA. PBMCs were activated in the presence of PHA and rhIL-2 for 3 days at 37°C as described. Pseudotyped HIV-1 virus or control medium without virus was added to the cells, followed by incubation for a further 3 days to allow infection. Cells were then treated with vehicle (EtOH) or 100 nM Dex or MPA for an additional 24 hrs. Acquisition and analysis was carried out as described in the methods . The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *, **, and *** indicate p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: HIV-1-mediated apoptosis is enhanced in the presence of Dex and MPA. PBMCs were activated in the presence of PHA and rhIL-2 for 3 days at 37°C as described. Pseudotyped HIV-1 virus or control medium without virus was added to the cells, followed by incubation for a further 3 days to allow infection. Cells were then treated with vehicle (EtOH) or 100 nM Dex or MPA for an additional 24 hrs. Acquisition and analysis was carried out as described in the methods . The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *, **, and *** indicate p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Incubation, Infection

    The GR is involved in GC- and Vpr-mediated apoptosis in CD4 + T-cells. PBMCs were treated with 100 nM Dex or 1 µM RU486 in the absence or presence of 5 µM Vpr peptide (amino acids 52–96) for 24 hrs. A tryptic BSA digest added at an equivalent mass/volume ratio of peptide, served as a control wherever Vpr peptide was present. Cells were obtained and stained as described in the methods . The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: The GR is involved in GC- and Vpr-mediated apoptosis in CD4 + T-cells. PBMCs were treated with 100 nM Dex or 1 µM RU486 in the absence or presence of 5 µM Vpr peptide (amino acids 52–96) for 24 hrs. A tryptic BSA digest added at an equivalent mass/volume ratio of peptide, served as a control wherever Vpr peptide was present. Cells were obtained and stained as described in the methods . The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Staining

    The GR is involved in MPA- and Vpr-mediated apoptosis in CD4 + T-cells. PBMCs were treated with vehicle (EtOH), 100 nM MPA, 1 µM RU486 or 100 nM MPA plus 1 µM RU486, in the absence (A) or presence (B) of 5 µM Vpr peptide for 24 hrs. Cells were stained and acquired by flow cytometry as indicated in the methods . In A cells were not incubated with balanced isotonic glucose-HEPES buffer while in B, this buffer was used and a tryptic BSA digest served as a control wherever Vpr peptide was not added, as described in Methods . The histograms show pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: The GR is involved in MPA- and Vpr-mediated apoptosis in CD4 + T-cells. PBMCs were treated with vehicle (EtOH), 100 nM MPA, 1 µM RU486 or 100 nM MPA plus 1 µM RU486, in the absence (A) or presence (B) of 5 µM Vpr peptide for 24 hrs. Cells were stained and acquired by flow cytometry as indicated in the methods . In A cells were not incubated with balanced isotonic glucose-HEPES buffer while in B, this buffer was used and a tryptic BSA digest served as a control wherever Vpr peptide was not added, as described in Methods . The histograms show pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Staining, Flow Cytometry, Cytometry, Incubation

    Apoptosis induction by Dex and MPA is most likely mediated primarily through the GR. (A) PBMCs were treated with vehicle (EtOH), 100 nM MPA, 10 nM Ald, 100 nM E2, 100 nM Mib, 100 nM R5020, 10 µM NET-A or 10 µM NET for 24 hrs at 37°C. Cells were surface stained with ant-CD3 and anti-CD4 antibodies, and apoptosis was detected using flow cytometry as described in Figure 1 . The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *** indicates p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: Apoptosis induction by Dex and MPA is most likely mediated primarily through the GR. (A) PBMCs were treated with vehicle (EtOH), 100 nM MPA, 10 nM Ald, 100 nM E2, 100 nM Mib, 100 nM R5020, 10 µM NET-A or 10 µM NET for 24 hrs at 37°C. Cells were surface stained with ant-CD3 and anti-CD4 antibodies, and apoptosis was detected using flow cytometry as described in Figure 1 . The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *** indicates p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Staining, Flow Cytometry, Cytometry

    GC and Vpr differentially regulate key genes involved in apoptosis. PBMCs were treated with or without 5 µM Vpr peptide as described previously and treated with or without 100 nM Dex, MPA, NET-A or P4 for 24 hrs. After treatment, RNA was extracted, reverse transcribed, and Bcl-2 (A) or, Bim (B) mRNA expression was measured by real time PCR, normalising to GAPDH expression levels. The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *, **, and *** indicate p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: GC and Vpr differentially regulate key genes involved in apoptosis. PBMCs were treated with or without 5 µM Vpr peptide as described previously and treated with or without 100 nM Dex, MPA, NET-A or P4 for 24 hrs. After treatment, RNA was extracted, reverse transcribed, and Bcl-2 (A) or, Bim (B) mRNA expression was measured by real time PCR, normalising to GAPDH expression levels. The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *, **, and *** indicate p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    The GR is involved in Vpr-mediated apoptosis in CD4 + T-cells. PBMCs were treated with 1 µM RU486 in the absence or presence of 5 µM Vpr peptide (amino acids 52–96) for 24 hrs. A tryptic BSA digest served as a control (bars 1 and 2) wherever Vpr peptide was not added and was added at an equivalent mass/volume of peptide. Cells were obtained and stained as described in the methods . The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: The GR is involved in Vpr-mediated apoptosis in CD4 + T-cells. PBMCs were treated with 1 µM RU486 in the absence or presence of 5 µM Vpr peptide (amino acids 52–96) for 24 hrs. A tryptic BSA digest served as a control (bars 1 and 2) wherever Vpr peptide was not added and was added at an equivalent mass/volume of peptide. Cells were obtained and stained as described in the methods . The histogram shows pooled results from two independent experiments with samples from three donors. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test or a paired t-test, where *, **, and *** indicate p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Staining

    Dose-dependent apoptosis induction with Dex, MPA, NET-A and P4 in CD4 + T-cells. PBMCs were treated with vehicle (EtOH), Dex, MPA, NET-A or P4 at the concentrations indicated for 24 hrs at 37°C. Cells were stained and analysed as described for Figure 1 . The figure shows pooled results from two independent experiments with samples from three donors. Error bars represent standard deviation. Statistical trend analysis for each dose response was performed by the Wilcox rank-sum test, as further extended by Cuzick [85] , and showed a significant trend only for Dex (p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: Dose-dependent apoptosis induction with Dex, MPA, NET-A and P4 in CD4 + T-cells. PBMCs were treated with vehicle (EtOH), Dex, MPA, NET-A or P4 at the concentrations indicated for 24 hrs at 37°C. Cells were stained and analysed as described for Figure 1 . The figure shows pooled results from two independent experiments with samples from three donors. Error bars represent standard deviation. Statistical trend analysis for each dose response was performed by the Wilcox rank-sum test, as further extended by Cuzick [85] , and showed a significant trend only for Dex (p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Staining, Standard Deviation

    The progestin MPA, but not NET-A or P4, induces apoptosis in CD4 + T-cells. Cells were treated with or without 100 nM Dex, 100 nM F, 1 µM MPA, 10 µM NET-A, 1 µM P4 or vehicle control (EtOH) for 24 hrs. Cells were stained with anti-CD4, anti-CD14, annexin V and 7-AAD using the Apoptosis Detection kit I (BD biosciences). (A) Gating strategy and representative zebra plots of untreated (EtOH), MPA or Dex treated PBMCs. (B) The histogram shows pooled results from two independent experiments with samples from three donors. Data were acquired on a FACS calibur system (BD Biosciences) and analyzed using Flo-Jo software (Tree Star Inc., San Carlos, CA, USA). Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *, **, and *** indicate p

    Journal: PLoS ONE

    Article Title: The Progestin-Only Contraceptive Medroxyprogesterone Acetate, but Not Norethisterone Acetate, Enhances HIV-1 Vpr-Mediated Apoptosis in Human CD4+ T Cells through the Glucocorticoid Receptor

    doi: 10.1371/journal.pone.0062895

    Figure Lengend Snippet: The progestin MPA, but not NET-A or P4, induces apoptosis in CD4 + T-cells. Cells were treated with or without 100 nM Dex, 100 nM F, 1 µM MPA, 10 µM NET-A, 1 µM P4 or vehicle control (EtOH) for 24 hrs. Cells were stained with anti-CD4, anti-CD14, annexin V and 7-AAD using the Apoptosis Detection kit I (BD biosciences). (A) Gating strategy and representative zebra plots of untreated (EtOH), MPA or Dex treated PBMCs. (B) The histogram shows pooled results from two independent experiments with samples from three donors. Data were acquired on a FACS calibur system (BD Biosciences) and analyzed using Flo-Jo software (Tree Star Inc., San Carlos, CA, USA). Statistical significance was determined by one-way ANOVA with Dunnett’s post-test, where *, **, and *** indicate p

    Article Snippet: Cell Treatment without Vpr Peptide and Flow Cytrometric Detection of Apoptosis Approximately 1×106 PBMCs/ml RPMI were seeded into a 5 mL Becton-Dickinson Falcon tube (352063).

    Techniques: Staining, FACS, Software

    MVAS, Tom70, and IRF3 mediate Sendai virus-induced apoptosis. (A) Wild-type (WT) and mavs −/− MEF cells were infected with SeV (MOI = 3) (left) or treated with TNF-α (10 ng/ml) and CHX (10 ng/ml) (right) for the indicated times.

    Journal: Journal of Virology

    Article Title: Tom70 Mediates Sendai Virus-Induced Apoptosis on Mitochondria

    doi: 10.1128/JVI.02959-14

    Figure Lengend Snippet: MVAS, Tom70, and IRF3 mediate Sendai virus-induced apoptosis. (A) Wild-type (WT) and mavs −/− MEF cells were infected with SeV (MOI = 3) (left) or treated with TNF-α (10 ng/ml) and CHX (10 ng/ml) (right) for the indicated times.

    Article Snippet: Cell apoptosis was detected using a FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed with FlowJo software (Ashland, OR).

    Techniques: Infection

    Schematic diagram of SeV-induced apoptosis. In resting cells, cytochrome c localizes to the mitochondrial intermembrane spaces, whereas Hsp90, IRF3, and Bax form a dynamic complex in the cytoplasm. (1) SeV triggers activation of the adapter MAVS on the

    Journal: Journal of Virology

    Article Title: Tom70 Mediates Sendai Virus-Induced Apoptosis on Mitochondria

    doi: 10.1128/JVI.02959-14

    Figure Lengend Snippet: Schematic diagram of SeV-induced apoptosis. In resting cells, cytochrome c localizes to the mitochondrial intermembrane spaces, whereas Hsp90, IRF3, and Bax form a dynamic complex in the cytoplasm. (1) SeV triggers activation of the adapter MAVS on the

    Article Snippet: Cell apoptosis was detected using a FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed with FlowJo software (Ashland, OR).

    Techniques: Activation Assay

    Tom70, Hsp90, and IRF3 form a dynamic complex during Sendai virus-induced apoptosis. (A, B, E, and G) HEK293T cells were transfected with the indicated constructs for 36 h and then subjected to immunoprecipitation with anti-HA antibody. The immunoprecipitates

    Journal: Journal of Virology

    Article Title: Tom70 Mediates Sendai Virus-Induced Apoptosis on Mitochondria

    doi: 10.1128/JVI.02959-14

    Figure Lengend Snippet: Tom70, Hsp90, and IRF3 form a dynamic complex during Sendai virus-induced apoptosis. (A, B, E, and G) HEK293T cells were transfected with the indicated constructs for 36 h and then subjected to immunoprecipitation with anti-HA antibody. The immunoprecipitates

    Article Snippet: Cell apoptosis was detected using a FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed with FlowJo software (Ashland, OR).

    Techniques: Transfection, Construct, Immunoprecipitation

    Sendai virus activates intrinsic mitochondrial apoptosis. (A and C) HEK293T cells were cotransfected with Bak and Bax (A) or Bcl-2 and Bax (C) for 36 h and then subjected to immunoprecipitation (IP) with anti-HA antibody. The immunoprecipitates were analyzed

    Journal: Journal of Virology

    Article Title: Tom70 Mediates Sendai Virus-Induced Apoptosis on Mitochondria

    doi: 10.1128/JVI.02959-14

    Figure Lengend Snippet: Sendai virus activates intrinsic mitochondrial apoptosis. (A and C) HEK293T cells were cotransfected with Bak and Bax (A) or Bcl-2 and Bax (C) for 36 h and then subjected to immunoprecipitation (IP) with anti-HA antibody. The immunoprecipitates were analyzed

    Article Snippet: Cell apoptosis was detected using a FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed with FlowJo software (Ashland, OR).

    Techniques: Immunoprecipitation

    Sendai virus-induced apoptosis depends on caspase-9. (A) HEK293 cells were infected with Sendai virus at an MOI of 0.4, 0.8, or 1.2 for 24 h (left) or at an MOI of 1 for 18, 21, or 24 h (right). Cell death was quantified using annexin V/PI double staining

    Journal: Journal of Virology

    Article Title: Tom70 Mediates Sendai Virus-Induced Apoptosis on Mitochondria

    doi: 10.1128/JVI.02959-14

    Figure Lengend Snippet: Sendai virus-induced apoptosis depends on caspase-9. (A) HEK293 cells were infected with Sendai virus at an MOI of 0.4, 0.8, or 1.2 for 24 h (left) or at an MOI of 1 for 18, 21, or 24 h (right). Cell death was quantified using annexin V/PI double staining

    Article Snippet: Cell apoptosis was detected using a FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed with FlowJo software (Ashland, OR).

    Techniques: Infection, Double Staining

    Sendai virus induces formation of a novel apoptosis complex (Tom70/Hsp90/IRF3/Bax). (A and B) (Left) HEK293T cells were transfected with the indicated plasmids for 36 h and then subjected to immunoprecipitation with anti-HA antibody. (Right) HEK293 cells

    Journal: Journal of Virology

    Article Title: Tom70 Mediates Sendai Virus-Induced Apoptosis on Mitochondria

    doi: 10.1128/JVI.02959-14

    Figure Lengend Snippet: Sendai virus induces formation of a novel apoptosis complex (Tom70/Hsp90/IRF3/Bax). (A and B) (Left) HEK293T cells were transfected with the indicated plasmids for 36 h and then subjected to immunoprecipitation with anti-HA antibody. (Right) HEK293 cells

    Article Snippet: Cell apoptosis was detected using a FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed with FlowJo software (Ashland, OR).

    Techniques: Transfection, Immunoprecipitation

    Inhibition of Hsp90 or IKK-i impairs Sendai virus-induced apoptosis. (A and B) HEK293 cells were treated with DMSO, RA (2.5 μM) (A), or GA (2 μM) (B) and then infected with SeV (left) or treated with TNF-α (10 ng/ml) and CHX (10

    Journal: Journal of Virology

    Article Title: Tom70 Mediates Sendai Virus-Induced Apoptosis on Mitochondria

    doi: 10.1128/JVI.02959-14

    Figure Lengend Snippet: Inhibition of Hsp90 or IKK-i impairs Sendai virus-induced apoptosis. (A and B) HEK293 cells were treated with DMSO, RA (2.5 μM) (A), or GA (2 μM) (B) and then infected with SeV (left) or treated with TNF-α (10 ng/ml) and CHX (10

    Article Snippet: Cell apoptosis was detected using a FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed with FlowJo software (Ashland, OR).

    Techniques: Inhibition, Infection

    IRF3 is recruited to mitochondria via Tom70/Hsp90 during Sendai virus-induced apoptosis. (A) Flag-IRF3 was ectopically expressed in HEK293 cells, which were then stimulated with SeV for the indicated times. Lysates of whole cells (W) and of mitochondria

    Journal: Journal of Virology

    Article Title: Tom70 Mediates Sendai Virus-Induced Apoptosis on Mitochondria

    doi: 10.1128/JVI.02959-14

    Figure Lengend Snippet: IRF3 is recruited to mitochondria via Tom70/Hsp90 during Sendai virus-induced apoptosis. (A) Flag-IRF3 was ectopically expressed in HEK293 cells, which were then stimulated with SeV for the indicated times. Lysates of whole cells (W) and of mitochondria

    Article Snippet: Cell apoptosis was detected using a FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed with FlowJo software (Ashland, OR).

    Techniques:

    The kinase activity of LKB1 is required to rescue AMPK-null MEF cells from AICAR-induced apoptosis A. H157 cells were treated with 1 mM AICAR, 1 μM A-769662 or their combination for 24 hrs. B. H157-LKB1-WT cells were pre-treated with 10 μM compound C followed by 1 mM AICAR treatment for 4 hrs. Nuclear (N) and cytosolic (C) fractions were analyzed by immunoblot with indicated antibodies. C. AMPK-null MEF cells were infected with retrovirus containing the indicated LKB1 constructs to obtain stable cell lines. NTR: N-terminal Region; K78M: Kinase-dead mutant. Cells were treated 0.25 mM uridine, 0.25 mM AICAR or their combination for 24 hrs. Cell lysates were analyzed by immunoblot with indicated antibodies, including the one against cleaved caspase-3.

    Journal: Oncotarget

    Article Title: LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions

    doi: 10.18632/oncotarget.6224

    Figure Lengend Snippet: The kinase activity of LKB1 is required to rescue AMPK-null MEF cells from AICAR-induced apoptosis A. H157 cells were treated with 1 mM AICAR, 1 μM A-769662 or their combination for 24 hrs. B. H157-LKB1-WT cells were pre-treated with 10 μM compound C followed by 1 mM AICAR treatment for 4 hrs. Nuclear (N) and cytosolic (C) fractions were analyzed by immunoblot with indicated antibodies. C. AMPK-null MEF cells were infected with retrovirus containing the indicated LKB1 constructs to obtain stable cell lines. NTR: N-terminal Region; K78M: Kinase-dead mutant. Cells were treated 0.25 mM uridine, 0.25 mM AICAR or their combination for 24 hrs. Cell lysates were analyzed by immunoblot with indicated antibodies, including the one against cleaved caspase-3.

    Article Snippet: Apoptosis analysis was carried out by FACS (BD Biosciences).

    Techniques: Activity Assay, Infection, Construct, Stable Transfection, Mutagenesis

    Depletion of uridine is responsible for AICAR-induced apoptosis in LKB1-null cells A. H460, H157 and Lkb1 −/− MEF cells were seeded in 96-well plates, and treated with the indicated concentration of uridine, cytidine, adenosine, thymidine, dTTP alone, or their respective combination with 1 mM AICAR. Plates were subjected to SRB assay 48 hrs after treatment. Reactions were carried out in quadruplicates, and the error bars represent one standard deviation. B. Lkb1 −/− MEF and H460 cells were seeded in 6-well plates and treated with 1 mM AICAR alone, 5 mM uridine alone or their combination. Lysates were collected 24 hrs after treatment for immunoblot analysis of total caspase-3. C. For Annexin-V and 7AAD analysis of apoptosis, Lkb1 −/− MEF and H460 cells were seeded in 6-well plates, and treated with 1 mM AICAR alone, 5 mM uridine alone, or their combination. Both floating and attached cells were collected 24 hrs after treatment and subjected to flow analysis. Percentage of Annexin-V positive cells was reported. D. Isogenic H157-pBabe and H157-LKB1-WT cells were treated with 0.2 mM of leflunomide for 48 hrs. Cell lysates were collected and analyzed by immunoblot with indicated antibodies. LKB1 was depleted in H1299 cells by siRNA and treated with 0, 0.1, or 0.2 mM of leflunomide for 48 hrs. Cell lysates were collected for immunblot of LKB1 and total caspase-3. E. H460 cells were treated with 0.25 mM phenformin, 25 μM leflunomide, or their combination for 10 days in a colony formation assay.

    Journal: Oncotarget

    Article Title: LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions

    doi: 10.18632/oncotarget.6224

    Figure Lengend Snippet: Depletion of uridine is responsible for AICAR-induced apoptosis in LKB1-null cells A. H460, H157 and Lkb1 −/− MEF cells were seeded in 96-well plates, and treated with the indicated concentration of uridine, cytidine, adenosine, thymidine, dTTP alone, or their respective combination with 1 mM AICAR. Plates were subjected to SRB assay 48 hrs after treatment. Reactions were carried out in quadruplicates, and the error bars represent one standard deviation. B. Lkb1 −/− MEF and H460 cells were seeded in 6-well plates and treated with 1 mM AICAR alone, 5 mM uridine alone or their combination. Lysates were collected 24 hrs after treatment for immunoblot analysis of total caspase-3. C. For Annexin-V and 7AAD analysis of apoptosis, Lkb1 −/− MEF and H460 cells were seeded in 6-well plates, and treated with 1 mM AICAR alone, 5 mM uridine alone, or their combination. Both floating and attached cells were collected 24 hrs after treatment and subjected to flow analysis. Percentage of Annexin-V positive cells was reported. D. Isogenic H157-pBabe and H157-LKB1-WT cells were treated with 0.2 mM of leflunomide for 48 hrs. Cell lysates were collected and analyzed by immunoblot with indicated antibodies. LKB1 was depleted in H1299 cells by siRNA and treated with 0, 0.1, or 0.2 mM of leflunomide for 48 hrs. Cell lysates were collected for immunblot of LKB1 and total caspase-3. E. H460 cells were treated with 0.25 mM phenformin, 25 μM leflunomide, or their combination for 10 days in a colony formation assay.

    Article Snippet: Apoptosis analysis was carried out by FACS (BD Biosciences).

    Techniques: Concentration Assay, Sulforhodamine B Assay, Standard Deviation, Flow Cytometry, Colony Assay

    Disruption of TIF-IA mediated pre-rRNA synthesis is responsible for AICAR-induced apoptosis in LKB1-null/depleted cells A. H460 and H157 cells were plated on glass cover slips and treated with 1 mM AICAR for 12 hrs. Cells were labeled with 2 mM 5-fluorouridine before immunofluorescence analysis. B. H460 and H157 cells were seeded in 60 mm dishes and treated with 1 mM AICAR, 2 mM uridine alone, or their combination. RNA was extracted with Trizol 12 hrs after AICAR treatment. Pre-RNA level was assessed by real-time PCR amplifying pre-45S ribosomal RNA using 18S rRNA as control. C. H460 and H157 cells were seeded in 6-well plates and treated with TIF-IA siRNA or control siRNA. Cell lysates were collected 48 hrs after transfection for immunoblot analysis and total caspase-3 was checked. D. H460 cells were stably infected with retrovirus containing the indicated TIF-IA wild-type or mutant constructs. Cells were treated with 1 mM AICAR for 24 hrs. Cell lysates were analyzed by immunoblot using indicated antibodies, including the one against cleaved caspase-3.

    Journal: Oncotarget

    Article Title: LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions

    doi: 10.18632/oncotarget.6224

    Figure Lengend Snippet: Disruption of TIF-IA mediated pre-rRNA synthesis is responsible for AICAR-induced apoptosis in LKB1-null/depleted cells A. H460 and H157 cells were plated on glass cover slips and treated with 1 mM AICAR for 12 hrs. Cells were labeled with 2 mM 5-fluorouridine before immunofluorescence analysis. B. H460 and H157 cells were seeded in 60 mm dishes and treated with 1 mM AICAR, 2 mM uridine alone, or their combination. RNA was extracted with Trizol 12 hrs after AICAR treatment. Pre-RNA level was assessed by real-time PCR amplifying pre-45S ribosomal RNA using 18S rRNA as control. C. H460 and H157 cells were seeded in 6-well plates and treated with TIF-IA siRNA or control siRNA. Cell lysates were collected 48 hrs after transfection for immunoblot analysis and total caspase-3 was checked. D. H460 cells were stably infected with retrovirus containing the indicated TIF-IA wild-type or mutant constructs. Cells were treated with 1 mM AICAR for 24 hrs. Cell lysates were analyzed by immunoblot using indicated antibodies, including the one against cleaved caspase-3.

    Article Snippet: Apoptosis analysis was carried out by FACS (BD Biosciences).

    Techniques: IA, Labeling, Immunofluorescence, Real-time Polymerase Chain Reaction, Transfection, Stable Transfection, Infection, Mutagenesis, Construct

    LKB1 promotes cell survival in uridine-depleted condition by maintaining pre-RNA synthesis TIF-IA-mediated pre-RNA synthesis is tightly regulated and genetic inactivation of TIF-IA is sufficient to promote apoptosis. AICAR treatment leads to the decrease of uridine-related metabolites. In LKB1-null cells, such decrease also results in the downregulation of pre-rRNA synthesis and cell death. Wild-type LKB1 is capable of maintaining pre-rRNA synthesis under these conditions to maintain cell survival.

    Journal: Oncotarget

    Article Title: LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions

    doi: 10.18632/oncotarget.6224

    Figure Lengend Snippet: LKB1 promotes cell survival in uridine-depleted condition by maintaining pre-RNA synthesis TIF-IA-mediated pre-RNA synthesis is tightly regulated and genetic inactivation of TIF-IA is sufficient to promote apoptosis. AICAR treatment leads to the decrease of uridine-related metabolites. In LKB1-null cells, such decrease also results in the downregulation of pre-rRNA synthesis and cell death. Wild-type LKB1 is capable of maintaining pre-rRNA synthesis under these conditions to maintain cell survival.

    Article Snippet: Apoptosis analysis was carried out by FACS (BD Biosciences).

    Techniques: IA

    AICAR treatment induces apoptosis specifically in LKB1-null NSCLC cells A. H460 isogenic cell lines were treated with 1 mM AICAR for 24 hrs. H157 and A549 isogenic cells were treated with 2 mM AICAR for 24 hrs and 48 hrs, respectively. K78M: kinase-dead mutant. B. H1299 cells were treated with LKB1 siRNA or control siRNA. 2 mM AICAR was added 48 hrs after siRNA transfection. C. H1299 and H1792 cells were treated with control lentivirus or virus containing LKB1-shRNA to obtain stable cell lines. Cells were treated with 2 mM AICAR for 48 hrs. D. LKB1-null MEF cells were infected with retrovirus containing indicated LKB1 constructs to obtain stable cell lines. NTR: N-terminal Region; KD: Kinase Domain; CTR: C-terminal Region; K78I: Kinase-Dead mutant; C430S: farnesylation motif mutation to inhibit farnesylation. Cells were treated with 1 mM AICAR for 24 hrs. In all experiments, cell lysates were collected after the indicated time of AICAR treatment for immunoblot analysis using indicated antibodies. Antibody against total caspase-3 was used in panel a, b, and c, and antibody against cleaved caspase-3 was used in panel d.

    Journal: Oncotarget

    Article Title: LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions

    doi: 10.18632/oncotarget.6224

    Figure Lengend Snippet: AICAR treatment induces apoptosis specifically in LKB1-null NSCLC cells A. H460 isogenic cell lines were treated with 1 mM AICAR for 24 hrs. H157 and A549 isogenic cells were treated with 2 mM AICAR for 24 hrs and 48 hrs, respectively. K78M: kinase-dead mutant. B. H1299 cells were treated with LKB1 siRNA or control siRNA. 2 mM AICAR was added 48 hrs after siRNA transfection. C. H1299 and H1792 cells were treated with control lentivirus or virus containing LKB1-shRNA to obtain stable cell lines. Cells were treated with 2 mM AICAR for 48 hrs. D. LKB1-null MEF cells were infected with retrovirus containing indicated LKB1 constructs to obtain stable cell lines. NTR: N-terminal Region; KD: Kinase Domain; CTR: C-terminal Region; K78I: Kinase-Dead mutant; C430S: farnesylation motif mutation to inhibit farnesylation. Cells were treated with 1 mM AICAR for 24 hrs. In all experiments, cell lysates were collected after the indicated time of AICAR treatment for immunoblot analysis using indicated antibodies. Antibody against total caspase-3 was used in panel a, b, and c, and antibody against cleaved caspase-3 was used in panel d.

    Article Snippet: Apoptosis analysis was carried out by FACS (BD Biosciences).

    Techniques: Mutagenesis, Transfection, shRNA, Stable Transfection, Infection, Construct

    The reverse signaling of tmTNF-α promotes AICD through upregulating FasL/Fas and TRAIL/DR4 (A, B, C, D) Jurkat cells were simultaneously stimulated with PHA-P (5 μg/ml) and TNF-α pAb (1:100) for 24 h. PHA-preactivated primary T cells were restimulated with αCD3 (10 μg/ml) and TNF-α pAb for 24 h. Expression of FasL/Fas (A, B) and TRAIL/DR4/DR5 (C, D) was detected by flow cytometry. (E, F) Jurkat cells transfected with 100 nM siRNA for FasL or TRAIL for 48 h were activated with PHA-P (5 μg/ml) in the absence or presence of TNF-α pAb for 24 h. The mRNA levels of FasL and TRAIL were determined by Real-time qPCR (E) and the apoptosis was detected by Annexin V/PI (F). All the quantitative data represent means ± S.D. of at least three independent experiments. * p

    Journal: Oncotarget

    Article Title: Transmembrane TNF-α promotes activation-induced cell death by forward and reverse signaling

    doi: 10.18632/oncotarget.19124

    Figure Lengend Snippet: The reverse signaling of tmTNF-α promotes AICD through upregulating FasL/Fas and TRAIL/DR4 (A, B, C, D) Jurkat cells were simultaneously stimulated with PHA-P (5 μg/ml) and TNF-α pAb (1:100) for 24 h. PHA-preactivated primary T cells were restimulated with αCD3 (10 μg/ml) and TNF-α pAb for 24 h. Expression of FasL/Fas (A, B) and TRAIL/DR4/DR5 (C, D) was detected by flow cytometry. (E, F) Jurkat cells transfected with 100 nM siRNA for FasL or TRAIL for 48 h were activated with PHA-P (5 μg/ml) in the absence or presence of TNF-α pAb for 24 h. The mRNA levels of FasL and TRAIL were determined by Real-time qPCR (E) and the apoptosis was detected by Annexin V/PI (F). All the quantitative data represent means ± S.D. of at least three independent experiments. * p

    Article Snippet: Reagents phytohemagglutinin (PHA-P) and Hoechst 33258 were purchased from Sigma-Aldrich; Mouse anti-human CD3 mAb(OKT-3, # 300314) was purchased from Biolegend; Recombinant Human sTNF-α was from Peprotech; Ni2 +-NTA-Agarose was from Clonetech; TAPI-1(TACE inhibitor, # sc-222337) was purchased from Santa Cruz Biotechnology; Human sTNF-α ELISA kit was from eBioscience; FITC Annexin V Apoptosis Detection kit was purchased from BD Pharmingen; Detoxi-GelTM Endotoxin Removing Columns (# 20344) was purchased from Thermo Fisher Scientific.

    Techniques: Expressing, Flow Cytometry, Cytometry, Transfection, Real-time Polymerase Chain Reaction

    tmTNF-α is involved in AICD (A, B, C, D) Jurkat cells transfected with 10 μM of TNF-α AS, TACE AS or NS for 48 h were treated with PHA-P (5 μg/ml) for 24 h. Apoptosis rate (A,C) was evaluated by Annexin V/PI and expression of tmTNF-α (A,C) and TACE (B) on the cell surface was analyzed by flow cytometry. Relative levels of TACE mRNA was detected by Real time qPCR (B). Western blot analysis of tmTNF-α expression (D). (E, F) PHA-preactivated primary T cells were transfected with 10 μM of TNF-α AS or NS for 48 h, and then treated with αCD3 (10 μg/ml) for 24 h. A TACE inhibitor TAPI-1 (10 μM) was added simultaneously with αCD3 treatment, and vehicle DMSO served as a control. Apoptosis was evaluated by Annexin V/PI and tmTNF-α expression was detected by flow cytometry. (G, H) tmTNF-α overexpressing 24 h-PHA activated and fixed Jurkat cells were cocultured with 3 h-PHA activated Jurkat cells at an effector/target ratio of 10:1 for 48 h (G). For primary T cells, tmTNF-α overexpressing, αCD3-restimulated and fixed T cells were co-cultured with PHA-preactivated T cells (preactivated T) at an effector/target ratio of 10:1 for 48h (H). For neutralization of tmTNF-α, effector cells were treated with TNF-α pAb for 30 min and then washed prior to the addition to the target cells. Apoptosis was determined by the Annexin V/PI. All the quantitative data represent means ± S.D. of at least three independent experiments. * p

    Journal: Oncotarget

    Article Title: Transmembrane TNF-α promotes activation-induced cell death by forward and reverse signaling

    doi: 10.18632/oncotarget.19124

    Figure Lengend Snippet: tmTNF-α is involved in AICD (A, B, C, D) Jurkat cells transfected with 10 μM of TNF-α AS, TACE AS or NS for 48 h were treated with PHA-P (5 μg/ml) for 24 h. Apoptosis rate (A,C) was evaluated by Annexin V/PI and expression of tmTNF-α (A,C) and TACE (B) on the cell surface was analyzed by flow cytometry. Relative levels of TACE mRNA was detected by Real time qPCR (B). Western blot analysis of tmTNF-α expression (D). (E, F) PHA-preactivated primary T cells were transfected with 10 μM of TNF-α AS or NS for 48 h, and then treated with αCD3 (10 μg/ml) for 24 h. A TACE inhibitor TAPI-1 (10 μM) was added simultaneously with αCD3 treatment, and vehicle DMSO served as a control. Apoptosis was evaluated by Annexin V/PI and tmTNF-α expression was detected by flow cytometry. (G, H) tmTNF-α overexpressing 24 h-PHA activated and fixed Jurkat cells were cocultured with 3 h-PHA activated Jurkat cells at an effector/target ratio of 10:1 for 48 h (G). For primary T cells, tmTNF-α overexpressing, αCD3-restimulated and fixed T cells were co-cultured with PHA-preactivated T cells (preactivated T) at an effector/target ratio of 10:1 for 48h (H). For neutralization of tmTNF-α, effector cells were treated with TNF-α pAb for 30 min and then washed prior to the addition to the target cells. Apoptosis was determined by the Annexin V/PI. All the quantitative data represent means ± S.D. of at least three independent experiments. * p

    Article Snippet: Reagents phytohemagglutinin (PHA-P) and Hoechst 33258 were purchased from Sigma-Aldrich; Mouse anti-human CD3 mAb(OKT-3, # 300314) was purchased from Biolegend; Recombinant Human sTNF-α was from Peprotech; Ni2 +-NTA-Agarose was from Clonetech; TAPI-1(TACE inhibitor, # sc-222337) was purchased from Santa Cruz Biotechnology; Human sTNF-α ELISA kit was from eBioscience; FITC Annexin V Apoptosis Detection kit was purchased from BD Pharmingen; Detoxi-GelTM Endotoxin Removing Columns (# 20344) was purchased from Thermo Fisher Scientific.

    Techniques: Transfection, Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Neutralization

    sTNF-α participates in AICD (A, B) Jurkat cells transfected with 10 μM of TNF-α AS or TACE AS for 48 h were stimulated with PHA-P (5 μg/ml) for 24 h. For primary T cells, PHA-preactivated T cells transfected with TNF-α AS for 48 h were restimulated with αCD3 (10 μg/ml) for 24 h; or PHA-preactivated T cells simultaneously treated with TAPI-1 (10 μM) and αCD3 (10 μg/ml) for 24 h. Concentration of sTNF-α in supernatants was detected by ELISA. (C, D) Recombinant human sTNF-α (50U/ml) was incubated with PHA-P activated Jurkat cells or αCD3-restimulated primary T cells for 24 h and the apoptosis was detected by Annexin V/PI. Non-stimulated Jurkat T cells or preactivated primary T cells served as a control. All the quantitative data represent means ± S.D. of at least three independent experiments. ** p

    Journal: Oncotarget

    Article Title: Transmembrane TNF-α promotes activation-induced cell death by forward and reverse signaling

    doi: 10.18632/oncotarget.19124

    Figure Lengend Snippet: sTNF-α participates in AICD (A, B) Jurkat cells transfected with 10 μM of TNF-α AS or TACE AS for 48 h were stimulated with PHA-P (5 μg/ml) for 24 h. For primary T cells, PHA-preactivated T cells transfected with TNF-α AS for 48 h were restimulated with αCD3 (10 μg/ml) for 24 h; or PHA-preactivated T cells simultaneously treated with TAPI-1 (10 μM) and αCD3 (10 μg/ml) for 24 h. Concentration of sTNF-α in supernatants was detected by ELISA. (C, D) Recombinant human sTNF-α (50U/ml) was incubated with PHA-P activated Jurkat cells or αCD3-restimulated primary T cells for 24 h and the apoptosis was detected by Annexin V/PI. Non-stimulated Jurkat T cells or preactivated primary T cells served as a control. All the quantitative data represent means ± S.D. of at least three independent experiments. ** p

    Article Snippet: Reagents phytohemagglutinin (PHA-P) and Hoechst 33258 were purchased from Sigma-Aldrich; Mouse anti-human CD3 mAb(OKT-3, # 300314) was purchased from Biolegend; Recombinant Human sTNF-α was from Peprotech; Ni2 +-NTA-Agarose was from Clonetech; TAPI-1(TACE inhibitor, # sc-222337) was purchased from Santa Cruz Biotechnology; Human sTNF-α ELISA kit was from eBioscience; FITC Annexin V Apoptosis Detection kit was purchased from BD Pharmingen; Detoxi-GelTM Endotoxin Removing Columns (# 20344) was purchased from Thermo Fisher Scientific.

    Techniques: Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation

    The reverse signaling of tmTNF-α enhances AICD (A, C) Jurkat cells were incubated with PHA-P (5 μg/ml), TNF-α pAb (1:100), sTNFR1 or with both PHA-P and TNF-α pAb, or PHA-P and sTNFR1 in indicated concentrations for 24 h. Apoptosis was detected by Annexin V/PI. (B) PHA preactivated primary T cells were stimulated with αCD3 (10 μg/ml), TNF-α pAb, or both for 24 h. Apoptosis was detected by Annexin V/PI. Rabbit serum served as a control. All the quantitative data represent means ± S.D. of at least three independent experiments. ** p

    Journal: Oncotarget

    Article Title: Transmembrane TNF-α promotes activation-induced cell death by forward and reverse signaling

    doi: 10.18632/oncotarget.19124

    Figure Lengend Snippet: The reverse signaling of tmTNF-α enhances AICD (A, C) Jurkat cells were incubated with PHA-P (5 μg/ml), TNF-α pAb (1:100), sTNFR1 or with both PHA-P and TNF-α pAb, or PHA-P and sTNFR1 in indicated concentrations for 24 h. Apoptosis was detected by Annexin V/PI. (B) PHA preactivated primary T cells were stimulated with αCD3 (10 μg/ml), TNF-α pAb, or both for 24 h. Apoptosis was detected by Annexin V/PI. Rabbit serum served as a control. All the quantitative data represent means ± S.D. of at least three independent experiments. ** p

    Article Snippet: Reagents phytohemagglutinin (PHA-P) and Hoechst 33258 were purchased from Sigma-Aldrich; Mouse anti-human CD3 mAb(OKT-3, # 300314) was purchased from Biolegend; Recombinant Human sTNF-α was from Peprotech; Ni2 +-NTA-Agarose was from Clonetech; TAPI-1(TACE inhibitor, # sc-222337) was purchased from Santa Cruz Biotechnology; Human sTNF-α ELISA kit was from eBioscience; FITC Annexin V Apoptosis Detection kit was purchased from BD Pharmingen; Detoxi-GelTM Endotoxin Removing Columns (# 20344) was purchased from Thermo Fisher Scientific.

    Techniques: Incubation

    The reverse signaling of tmTNF-α enhances the sensitivity of T cells to TNF-α-induced AICD through upregulating TNFR (A, B, C, D) Jurkat cells were stimulated with PHA-P (5 μg/ml) in the absence or presence of TNF-α pAb (1:100) for 24 h. PHA preactivated T cells were reactivated by αCD3 (10 μg/ml) with or without TNF-α pAb (1:100) for 24 h. The expression of tmTNF-α (A, B) and two types of TNFR (C, D) was detected by flow cytometry. (E, F) Jurkat cells activated for 3 h by PHA-P (5 μg/ml) with or without TNF-α pAb were incubated for 4 h with sTNF-α (50 U/ml) or tmTNF-α stably transfected and fixed NIH3T3 cells at an effector/target ratio of 10:1. The apoptosis was detected by Annexin V/PI. (G, H, I) Jurkat cells transfected with 100 nM siRNA for TNFR1 or TNFR2 for 48 h were activated by PHA-P (5 μg/ml) with TNF-α pAb for 3 h, followed by incubation for 4 h with sTNF-α (50 U/ml) or tmTNF-α stably transfected and fixed NIH3T3 cells at an effector/target ratio of 10:1. The expression of TNFR1 and TNFR2 was analyzed by flow cytometry (G) and the apoptosis was detected by Annexin V/PI (H, I). For neutralization of tmTNF-α, the effector cells were treated with TNF-α pAb for 30 min prior to the addition to the target cells. All the quantitative data represent means ± S.D. of at least three independent experiments. * p

    Journal: Oncotarget

    Article Title: Transmembrane TNF-α promotes activation-induced cell death by forward and reverse signaling

    doi: 10.18632/oncotarget.19124

    Figure Lengend Snippet: The reverse signaling of tmTNF-α enhances the sensitivity of T cells to TNF-α-induced AICD through upregulating TNFR (A, B, C, D) Jurkat cells were stimulated with PHA-P (5 μg/ml) in the absence or presence of TNF-α pAb (1:100) for 24 h. PHA preactivated T cells were reactivated by αCD3 (10 μg/ml) with or without TNF-α pAb (1:100) for 24 h. The expression of tmTNF-α (A, B) and two types of TNFR (C, D) was detected by flow cytometry. (E, F) Jurkat cells activated for 3 h by PHA-P (5 μg/ml) with or without TNF-α pAb were incubated for 4 h with sTNF-α (50 U/ml) or tmTNF-α stably transfected and fixed NIH3T3 cells at an effector/target ratio of 10:1. The apoptosis was detected by Annexin V/PI. (G, H, I) Jurkat cells transfected with 100 nM siRNA for TNFR1 or TNFR2 for 48 h were activated by PHA-P (5 μg/ml) with TNF-α pAb for 3 h, followed by incubation for 4 h with sTNF-α (50 U/ml) or tmTNF-α stably transfected and fixed NIH3T3 cells at an effector/target ratio of 10:1. The expression of TNFR1 and TNFR2 was analyzed by flow cytometry (G) and the apoptosis was detected by Annexin V/PI (H, I). For neutralization of tmTNF-α, the effector cells were treated with TNF-α pAb for 30 min prior to the addition to the target cells. All the quantitative data represent means ± S.D. of at least three independent experiments. * p

    Article Snippet: Reagents phytohemagglutinin (PHA-P) and Hoechst 33258 were purchased from Sigma-Aldrich; Mouse anti-human CD3 mAb(OKT-3, # 300314) was purchased from Biolegend; Recombinant Human sTNF-α was from Peprotech; Ni2 +-NTA-Agarose was from Clonetech; TAPI-1(TACE inhibitor, # sc-222337) was purchased from Santa Cruz Biotechnology; Human sTNF-α ELISA kit was from eBioscience; FITC Annexin V Apoptosis Detection kit was purchased from BD Pharmingen; Detoxi-GelTM Endotoxin Removing Columns (# 20344) was purchased from Thermo Fisher Scientific.

    Techniques: Expressing, Flow Cytometry, Cytometry, Incubation, Stable Transfection, Transfection, Neutralization

    Elevation of tmTNF-α expression in AICD (A) Jurkat cells were activated with PHA-P for 24 h in indicated doses and the apoptosis was evaluated by Annexin V/PI. (B, C, D) Jurkat cells were stimulated with 5 μg/ml PHA for indicated time points. The apoptosis (B) and tmTNF-α expression (C) were analyzed by flow cytometry. The kinetic curves of apoptosis rate and tmTNF-α expression were shown in the panel D. (E, F, G) Primary human T cells were preactivated by PHA (5 μg/ml) for 3 days and cultured in the presence of IL-2 (50 U/ml) for 7 days. The preactivated T cells were restimulated for 24 h with plate-coated αCD3 (OKT3, 10 μg/ml). The apoptosis was measured by Annexin V/PI (E, left) and its quantitative analysis was shown in the histogram (E, right). Hochest/PI double staining in situ for apoptosis (F). The tmTNF-α expression was determined by flow cytometry (G, left) and its quantitative analysis was shown in the histogram (G, right). All the quantitative data represent means ± S.D. of at least three independent experiments. * p

    Journal: Oncotarget

    Article Title: Transmembrane TNF-α promotes activation-induced cell death by forward and reverse signaling

    doi: 10.18632/oncotarget.19124

    Figure Lengend Snippet: Elevation of tmTNF-α expression in AICD (A) Jurkat cells were activated with PHA-P for 24 h in indicated doses and the apoptosis was evaluated by Annexin V/PI. (B, C, D) Jurkat cells were stimulated with 5 μg/ml PHA for indicated time points. The apoptosis (B) and tmTNF-α expression (C) were analyzed by flow cytometry. The kinetic curves of apoptosis rate and tmTNF-α expression were shown in the panel D. (E, F, G) Primary human T cells were preactivated by PHA (5 μg/ml) for 3 days and cultured in the presence of IL-2 (50 U/ml) for 7 days. The preactivated T cells were restimulated for 24 h with plate-coated αCD3 (OKT3, 10 μg/ml). The apoptosis was measured by Annexin V/PI (E, left) and its quantitative analysis was shown in the histogram (E, right). Hochest/PI double staining in situ for apoptosis (F). The tmTNF-α expression was determined by flow cytometry (G, left) and its quantitative analysis was shown in the histogram (G, right). All the quantitative data represent means ± S.D. of at least three independent experiments. * p

    Article Snippet: Reagents phytohemagglutinin (PHA-P) and Hoechst 33258 were purchased from Sigma-Aldrich; Mouse anti-human CD3 mAb(OKT-3, # 300314) was purchased from Biolegend; Recombinant Human sTNF-α was from Peprotech; Ni2 +-NTA-Agarose was from Clonetech; TAPI-1(TACE inhibitor, # sc-222337) was purchased from Santa Cruz Biotechnology; Human sTNF-α ELISA kit was from eBioscience; FITC Annexin V Apoptosis Detection kit was purchased from BD Pharmingen; Detoxi-GelTM Endotoxin Removing Columns (# 20344) was purchased from Thermo Fisher Scientific.

    Techniques: Expressing, Flow Cytometry, Cytometry, Cell Culture, Double Staining, In Situ

    IκBSR attenuates the anti-apoptotic activity of ANG. (A) Western blotting analysis of IκB-α protein level in the vector control and in IκBSR transfectants. (B, C) Flow cytometric analyses of apoptotic cells in vector control transfectants (B) and in IκBSR transfectants (C). Cells were cultured in serum-free medium in the absence or presence of ANG (0.5 µg·mL −1 ) for 18 h, and analyzed using the FITC–Annexin V Apoptosis Detection Kit I. Cells cultured in 10% fetal bovine serum were used to set the cut-off value of propidium iodide and Annexin–FITC staining.

    Journal: The FEBS journal

    Article Title: Angiogenin prevents serum withdrawal-induced apoptosis of P19 embryonal carcinoma cells

    doi: 10.1111/j.1742-4658.2010.07766.x

    Figure Lengend Snippet: IκBSR attenuates the anti-apoptotic activity of ANG. (A) Western blotting analysis of IκB-α protein level in the vector control and in IκBSR transfectants. (B, C) Flow cytometric analyses of apoptotic cells in vector control transfectants (B) and in IκBSR transfectants (C). Cells were cultured in serum-free medium in the absence or presence of ANG (0.5 µg·mL −1 ) for 18 h, and analyzed using the FITC–Annexin V Apoptosis Detection Kit I. Cells cultured in 10% fetal bovine serum were used to set the cut-off value of propidium iodide and Annexin–FITC staining.

    Article Snippet: Transfected cells were cultured in serum-free medium in the absence or presence of 0.5 µg·mL−1 ANG for 18 h. Apoptotic cells were detected by flow cytometric analysis using an FITC–Annexin V Apoptosis Detection Kit I (BD Pharmingen).

    Techniques: Activity Assay, Western Blot, Plasmid Preparation, Flow Cytometry, Cell Culture, Staining

    ANG prevents serum withdrawal-induced apoptosis of P19 cells. (A) DNA fragmentation analysis. Cells were cultured in 10% fetal bovine serum (FBS) or in serum-free medium with the indicated concentration of ANG for 18 h. DNA was extracted and analyzed using the Apoptotic DNA Ladder Kit. The positive control included in the kit was the DNA fragments from apoptotic U937 cells. (B) Flow cytometry of FITC–Annexin. Cells were cultured in serum-free medium in the absence or presence of ANG (0.5 µg·mL −1 ) for 18 h, and analyzed using the FITC–Annexin V Apoptosis Detection Kit I. Cells cultured in 10% fetal bovine serum were used to set the cut-off value of propidium iodide and Annexin–FITC staining. (C) EB/AO staining. Cells were cultured in 10% fetal bovine serum or in serum-free medium in the absence or presence of ANG for 18 h. Cells were collected, stained with EB/AO and applied to a microscope slide for imaging. EB-stained (apoptotic) cells were counted in a total of 750 cells from five randomly selected areas of each slide, and the percentage of apoptotic cells is shown at the bottom of the image. The data shown are the means and standard errors of triplicates of a representative experiment of at least three repeats.

    Journal: The FEBS journal

    Article Title: Angiogenin prevents serum withdrawal-induced apoptosis of P19 embryonal carcinoma cells

    doi: 10.1111/j.1742-4658.2010.07766.x

    Figure Lengend Snippet: ANG prevents serum withdrawal-induced apoptosis of P19 cells. (A) DNA fragmentation analysis. Cells were cultured in 10% fetal bovine serum (FBS) or in serum-free medium with the indicated concentration of ANG for 18 h. DNA was extracted and analyzed using the Apoptotic DNA Ladder Kit. The positive control included in the kit was the DNA fragments from apoptotic U937 cells. (B) Flow cytometry of FITC–Annexin. Cells were cultured in serum-free medium in the absence or presence of ANG (0.5 µg·mL −1 ) for 18 h, and analyzed using the FITC–Annexin V Apoptosis Detection Kit I. Cells cultured in 10% fetal bovine serum were used to set the cut-off value of propidium iodide and Annexin–FITC staining. (C) EB/AO staining. Cells were cultured in 10% fetal bovine serum or in serum-free medium in the absence or presence of ANG for 18 h. Cells were collected, stained with EB/AO and applied to a microscope slide for imaging. EB-stained (apoptotic) cells were counted in a total of 750 cells from five randomly selected areas of each slide, and the percentage of apoptotic cells is shown at the bottom of the image. The data shown are the means and standard errors of triplicates of a representative experiment of at least three repeats.

    Article Snippet: Transfected cells were cultured in serum-free medium in the absence or presence of 0.5 µg·mL−1 ANG for 18 h. Apoptotic cells were detected by flow cytometric analysis using an FITC–Annexin V Apoptosis Detection Kit I (BD Pharmingen).

    Techniques: Cell Culture, Concentration Assay, Positive Control, Flow Cytometry, Cytometry, Staining, Microscopy, Imaging

    Bcl-2 siRNA inhibits the protective activity of ANG. (A) Western blotting analysis of Bcl-2 protein level in vector control and in Bcl-2-specific shRNA transfectants. The bar graph below the western panel is the relative density of Bcl-2 with β-actin as the normalization control. (B, C) Flow cytometric analyses of apoptotic cells in vector control transfectants (B) and in Bcl-2 shRNA transfectants (C). Cells were cultured in serum-free medium in the absence or presence of ANG (0.5 µg·mL −1 ) for 18 h, and analyzed using the FITC–Annexin V Apoptosis Detection Kit I. Cells cultured in 10% fetal bovine serum were used to set the cut-off value of propidium iodide and Annexin–FITC staining.

    Journal: The FEBS journal

    Article Title: Angiogenin prevents serum withdrawal-induced apoptosis of P19 embryonal carcinoma cells

    doi: 10.1111/j.1742-4658.2010.07766.x

    Figure Lengend Snippet: Bcl-2 siRNA inhibits the protective activity of ANG. (A) Western blotting analysis of Bcl-2 protein level in vector control and in Bcl-2-specific shRNA transfectants. The bar graph below the western panel is the relative density of Bcl-2 with β-actin as the normalization control. (B, C) Flow cytometric analyses of apoptotic cells in vector control transfectants (B) and in Bcl-2 shRNA transfectants (C). Cells were cultured in serum-free medium in the absence or presence of ANG (0.5 µg·mL −1 ) for 18 h, and analyzed using the FITC–Annexin V Apoptosis Detection Kit I. Cells cultured in 10% fetal bovine serum were used to set the cut-off value of propidium iodide and Annexin–FITC staining.

    Article Snippet: Transfected cells were cultured in serum-free medium in the absence or presence of 0.5 µg·mL−1 ANG for 18 h. Apoptotic cells were detected by flow cytometric analysis using an FITC–Annexin V Apoptosis Detection Kit I (BD Pharmingen).

    Techniques: Activity Assay, Western Blot, Plasmid Preparation, shRNA, Flow Cytometry, Cell Culture, Staining

    Cisplatin treatment in PKCδ silenced A375 human melanoma cells induces ceramide mediated apoptosis through IRF1-TNFα axis (A) 2 × 10 6 A375 cells were transfected with PKCδ specific siRNA or control siRNA as mentioned in Materials and Methods and the transfected cells were collected in TRIZOL for mRNA expression of PKCδ by semi quantitative RT-PCR. GAPDH was used as a reference. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P

    Journal: Oncotarget

    Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

    doi: 10.18632/oncotarget.26478

    Figure Lengend Snippet: Cisplatin treatment in PKCδ silenced A375 human melanoma cells induces ceramide mediated apoptosis through IRF1-TNFα axis (A) 2 × 10 6 A375 cells were transfected with PKCδ specific siRNA or control siRNA as mentioned in Materials and Methods and the transfected cells were collected in TRIZOL for mRNA expression of PKCδ by semi quantitative RT-PCR. GAPDH was used as a reference. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P

    Article Snippet: In vitro tumor apoptosis assay In vitro apoptosis of tumor cells was determined using the apoptosis detection kit (BD Pharmingen) as per the manufacturer's protocol.

    Techniques: Transfection, Expressing, Quantitative RT-PCR

    Diagrammatic representation of cisplatin mediated apoptosis of B16F10 melanoma cells in PKCδ independent manner

    Journal: Oncotarget

    Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

    doi: 10.18632/oncotarget.26478

    Figure Lengend Snippet: Diagrammatic representation of cisplatin mediated apoptosis of B16F10 melanoma cells in PKCδ independent manner

    Article Snippet: In vitro tumor apoptosis assay In vitro apoptosis of tumor cells was determined using the apoptosis detection kit (BD Pharmingen) as per the manufacturer's protocol.

    Techniques:

    Cisplatin induces apoptosis in PKCδ silenced B16F10 cells by TNFα mediated pathway (A) and (B) B16F10 cells were transfected with PKCδ siRNA followed by cisplatin, cisplatin along with impiramine, and C2 ceramide treatment. The treated cells were collected in TRIZOL for mRNA expression analyses of TNF-α, IL-12, IFN-γ, IL-10 and TGF-β by semi quantitative RT-PCR. The cell supernatants were collected for estimation of TNF-α, IL-12, IFN-γ, IL-10 and TGF-β by ELISA. GAPDH was used as a reference. Data are from one of three representative experiments. Bar diagram is represented as mean ± SD. * P

    Journal: Oncotarget

    Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

    doi: 10.18632/oncotarget.26478

    Figure Lengend Snippet: Cisplatin induces apoptosis in PKCδ silenced B16F10 cells by TNFα mediated pathway (A) and (B) B16F10 cells were transfected with PKCδ siRNA followed by cisplatin, cisplatin along with impiramine, and C2 ceramide treatment. The treated cells were collected in TRIZOL for mRNA expression analyses of TNF-α, IL-12, IFN-γ, IL-10 and TGF-β by semi quantitative RT-PCR. The cell supernatants were collected for estimation of TNF-α, IL-12, IFN-γ, IL-10 and TGF-β by ELISA. GAPDH was used as a reference. Data are from one of three representative experiments. Bar diagram is represented as mean ± SD. * P

    Article Snippet: In vitro tumor apoptosis assay In vitro apoptosis of tumor cells was determined using the apoptosis detection kit (BD Pharmingen) as per the manufacturer's protocol.

    Techniques: Transfection, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Cisplatin induces IRF-1 dependent TNFα activation for initiation of apoptosis in PKCδ silenced B16F10 melanoma cells (A) and (B) B16F10 cells were transfected with PKCδ siRNA followed by Cisplatin treatment. The treated cells were collected in TRIZOL for mRNA extraction and semi quantitative RT-PCR analyses for IRF 1- 8 were done. The expression IRF 1 was analyzed in whole cell lysates by Western blotting. GAPDH was used as a reference. Data are from one of three representative experiments. Bar diagram is represented as mean ± SD. * P

    Journal: Oncotarget

    Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

    doi: 10.18632/oncotarget.26478

    Figure Lengend Snippet: Cisplatin induces IRF-1 dependent TNFα activation for initiation of apoptosis in PKCδ silenced B16F10 melanoma cells (A) and (B) B16F10 cells were transfected with PKCδ siRNA followed by Cisplatin treatment. The treated cells were collected in TRIZOL for mRNA extraction and semi quantitative RT-PCR analyses for IRF 1- 8 were done. The expression IRF 1 was analyzed in whole cell lysates by Western blotting. GAPDH was used as a reference. Data are from one of three representative experiments. Bar diagram is represented as mean ± SD. * P

    Article Snippet: In vitro tumor apoptosis assay In vitro apoptosis of tumor cells was determined using the apoptosis detection kit (BD Pharmingen) as per the manufacturer's protocol.

    Techniques: Activation Assay, Transfection, Quantitative RT-PCR, Expressing, Western Blot

    cPLA 2 modulates cisplatin mediated apoptosis in PKCδ silenced B16F10 melanoma cells (A) and (B) B16F10 cells were transfected with PKCδ siRNA followed by Cisplatin treatment. cPLA 2 activity was determined from cell lysates using the cytosolic phospholipase A2 assay kit according to the manufacturer's instructions. Bar diagram is represented as mean ± SD. * P

    Journal: Oncotarget

    Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

    doi: 10.18632/oncotarget.26478

    Figure Lengend Snippet: cPLA 2 modulates cisplatin mediated apoptosis in PKCδ silenced B16F10 melanoma cells (A) and (B) B16F10 cells were transfected with PKCδ siRNA followed by Cisplatin treatment. cPLA 2 activity was determined from cell lysates using the cytosolic phospholipase A2 assay kit according to the manufacturer's instructions. Bar diagram is represented as mean ± SD. * P

    Article Snippet: In vitro tumor apoptosis assay In vitro apoptosis of tumor cells was determined using the apoptosis detection kit (BD Pharmingen) as per the manufacturer's protocol.

    Techniques: Transfection, Activity Assay

    Cisplatin continues to induce apoptosis by ceramide generation during hypoxic condition in PKCδ silenced B16F10 Cells (A) and (B) B16F10 cells transfected with PKCδ siRNA followed by cisplatin treatment were cultured in hypoxic chamber as described in Materials and Methods. Cells were collected and subsequently stained with anti-mouse ceramide-FITC, isotype-matched control mouse antibody. Ceramide expression was measured by flow cytometry analysis. In a different experiment as mentioned in Materials and Methods, cells were stained with Annexin-V FITC and PI and subsequently analyzed by flow cytometry. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P

    Journal: Oncotarget

    Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

    doi: 10.18632/oncotarget.26478

    Figure Lengend Snippet: Cisplatin continues to induce apoptosis by ceramide generation during hypoxic condition in PKCδ silenced B16F10 Cells (A) and (B) B16F10 cells transfected with PKCδ siRNA followed by cisplatin treatment were cultured in hypoxic chamber as described in Materials and Methods. Cells were collected and subsequently stained with anti-mouse ceramide-FITC, isotype-matched control mouse antibody. Ceramide expression was measured by flow cytometry analysis. In a different experiment as mentioned in Materials and Methods, cells were stained with Annexin-V FITC and PI and subsequently analyzed by flow cytometry. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P

    Article Snippet: In vitro tumor apoptosis assay In vitro apoptosis of tumor cells was determined using the apoptosis detection kit (BD Pharmingen) as per the manufacturer's protocol.

    Techniques: Transfection, Cell Culture, Staining, Expressing, Flow Cytometry, Cytometry

    Cisplatin inhibits cell cycle progression and induces apoptosis in PKCδ silenced B16F10 cells via ceramide generation (A) 2 × 10 6 B16F10 cells were transfected with PKCδ specific siRNA or control siRNA as mentioned in Materials and Methods. The transfected cells were collected in TRIZOL for mRNA expression of PKCδ by semi quantitative RT-PCR. GAPDH was used as a reference. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P

    Journal: Oncotarget

    Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

    doi: 10.18632/oncotarget.26478

    Figure Lengend Snippet: Cisplatin inhibits cell cycle progression and induces apoptosis in PKCδ silenced B16F10 cells via ceramide generation (A) 2 × 10 6 B16F10 cells were transfected with PKCδ specific siRNA or control siRNA as mentioned in Materials and Methods. The transfected cells were collected in TRIZOL for mRNA expression of PKCδ by semi quantitative RT-PCR. GAPDH was used as a reference. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P

    Article Snippet: In vitro tumor apoptosis assay In vitro apoptosis of tumor cells was determined using the apoptosis detection kit (BD Pharmingen) as per the manufacturer's protocol.

    Techniques: Transfection, Expressing, Quantitative RT-PCR

    Bcl-2 cannot block apoptosis induced by c-Bax. a Differential expression of Bax-HA and ΔBax-HA. AGS cells were transfected with 1 µg, 2 µg, or 4 µg of either Bax-HA or ΔBax-HA for 24 h and subjected to immunoblotting for protein expression. b Short-lived ΔBax-HA than Bax-HA. CHX (50 ug/ml) was added to 293 T or AGS cells transfected with plasmid encoding Bax-HA or ΔBax-HA prior to treatment and cultured for 24 h. Equivalent lysates from the cells harvested at the indicated time points were immunoblotted for indicated protein expression. c , d , g Bcl-2 did not decrease apoptosis induced by ΔBax-HA. 1 µg Bax-HA or 4 µg ΔBax-HA plasmid was transfected with 4 µg Bcl-2-Flag ( c ) or 4 µg ΔBax-HA and indicated quality of Bcl-2-Flag plasmid, then cultured for 24 h and AGS subjected to crystal violet staining or immunoblot ( c , g ). AGS cells were treated with 1 µg Bax-HA or 4 µg ΔBax-HA plasmid ( d ) and subjected to Annexin V/propidium iodide double staining and FCM to detect apoptosis. e , f Cleaved Bax was a stronger apoptosis inducer. e The tet-on system was constructed and protein expression was induced by 10uM Tetracyclines for 24 h in AGS. Apoptosis level and protein level was detected by FCM and immunoblot. f The AGS stable cell line was constructed and incubated with 50 nM MLN8237 for 72 h. Apoptosis level and protein level was detected by FCM and immunoblot. Error bars represent SD from three independent experiments. This immunoblot is representative of three independent experiments. Asterisk (*) indicates a significant difference. **** P

    Journal: Cell Death & Disease

    Article Title: Suppression of AURKA alleviates p27 inhibition on Bax cleavage and induces more intensive apoptosis in gastric cancer

    doi: 10.1038/s41419-018-0823-3

    Figure Lengend Snippet: Bcl-2 cannot block apoptosis induced by c-Bax. a Differential expression of Bax-HA and ΔBax-HA. AGS cells were transfected with 1 µg, 2 µg, or 4 µg of either Bax-HA or ΔBax-HA for 24 h and subjected to immunoblotting for protein expression. b Short-lived ΔBax-HA than Bax-HA. CHX (50 ug/ml) was added to 293 T or AGS cells transfected with plasmid encoding Bax-HA or ΔBax-HA prior to treatment and cultured for 24 h. Equivalent lysates from the cells harvested at the indicated time points were immunoblotted for indicated protein expression. c , d , g Bcl-2 did not decrease apoptosis induced by ΔBax-HA. 1 µg Bax-HA or 4 µg ΔBax-HA plasmid was transfected with 4 µg Bcl-2-Flag ( c ) or 4 µg ΔBax-HA and indicated quality of Bcl-2-Flag plasmid, then cultured for 24 h and AGS subjected to crystal violet staining or immunoblot ( c , g ). AGS cells were treated with 1 µg Bax-HA or 4 µg ΔBax-HA plasmid ( d ) and subjected to Annexin V/propidium iodide double staining and FCM to detect apoptosis. e , f Cleaved Bax was a stronger apoptosis inducer. e The tet-on system was constructed and protein expression was induced by 10uM Tetracyclines for 24 h in AGS. Apoptosis level and protein level was detected by FCM and immunoblot. f The AGS stable cell line was constructed and incubated with 50 nM MLN8237 for 72 h. Apoptosis level and protein level was detected by FCM and immunoblot. Error bars represent SD from three independent experiments. This immunoblot is representative of three independent experiments. Asterisk (*) indicates a significant difference. **** P

    Article Snippet: To measure apoptosis, mitochondrial membrane potential and Calcium influxes, cells were harvested and stained with FITC Apoptosis Detection Dye, JC-1, Fluo-3 AM (Beyotime Biotechnology), and then analyzed by flow cytometry.

    Techniques: Blocking Assay, Expressing, Transfection, Plasmid Preparation, Cell Culture, Staining, Double Staining, Construct, Stable Transfection, Incubation

    AURKA and p27 were overexpressed in tumor tissues and had a positive correlation in late TNM stage of gastric cancer tissues. a – d IHC staining of human gastric tumor tissues and adjacent tissues using specific AURKA and p27 antibodies, respectively. Samples were classified according to the intensity of the staining of AURKA and p27 expression. e A four-fold square showed the correlations of AURKA and p27 expression in gastric cancer. f Working model of the suppression of AURKA in gastric cancer cells relieved inhibition of Bax cleavage by p27 and induced more intensive apoptosis

    Journal: Cell Death & Disease

    Article Title: Suppression of AURKA alleviates p27 inhibition on Bax cleavage and induces more intensive apoptosis in gastric cancer

    doi: 10.1038/s41419-018-0823-3

    Figure Lengend Snippet: AURKA and p27 were overexpressed in tumor tissues and had a positive correlation in late TNM stage of gastric cancer tissues. a – d IHC staining of human gastric tumor tissues and adjacent tissues using specific AURKA and p27 antibodies, respectively. Samples were classified according to the intensity of the staining of AURKA and p27 expression. e A four-fold square showed the correlations of AURKA and p27 expression in gastric cancer. f Working model of the suppression of AURKA in gastric cancer cells relieved inhibition of Bax cleavage by p27 and induced more intensive apoptosis

    Article Snippet: To measure apoptosis, mitochondrial membrane potential and Calcium influxes, cells were harvested and stained with FITC Apoptosis Detection Dye, JC-1, Fluo-3 AM (Beyotime Biotechnology), and then analyzed by flow cytometry.

    Techniques: Immunohistochemistry, Staining, Expressing, Inhibition

    Cell cytotoxicity of gastric cancer cells induced by MLN8237 treatment. a , b MLN8237 suppressed the proliferation of gastric cancer cells. AGS, SGC-7901, NCI-N87 and KATO III cells were treated with MLN8237 at the indicated concentrations for 24–96 h, then counted cell number a with MTS assay or b with Hoechst 33342 staining in AGS cell line. The mean and SDs of the plots were obtained from three wells within three independent assays. c MLN8237 treatment induced apoptosis of AGS cell line as determined by Annexin V/propidium iodide double staining and FCM. AGS was treatment with MLN8237 for 72 h. Statistical analysis from triplicates of different biological experiments. d MLN8237 treatment induced G2/M arrest and polyploidy in gastric cancer cells. The cell cycle profile was determined by treating AGS cells with the indicated concentration of MLN8237 for 72 h, staining the cells with propidium iodide staining and analyzing by FCM. This staining is representative of three different independent experiments. This immunoblot is representative of three independent experiments. Asterisk (*) indicates a significant difference. *** P

    Journal: Cell Death & Disease

    Article Title: Suppression of AURKA alleviates p27 inhibition on Bax cleavage and induces more intensive apoptosis in gastric cancer

    doi: 10.1038/s41419-018-0823-3

    Figure Lengend Snippet: Cell cytotoxicity of gastric cancer cells induced by MLN8237 treatment. a , b MLN8237 suppressed the proliferation of gastric cancer cells. AGS, SGC-7901, NCI-N87 and KATO III cells were treated with MLN8237 at the indicated concentrations for 24–96 h, then counted cell number a with MTS assay or b with Hoechst 33342 staining in AGS cell line. The mean and SDs of the plots were obtained from three wells within three independent assays. c MLN8237 treatment induced apoptosis of AGS cell line as determined by Annexin V/propidium iodide double staining and FCM. AGS was treatment with MLN8237 for 72 h. Statistical analysis from triplicates of different biological experiments. d MLN8237 treatment induced G2/M arrest and polyploidy in gastric cancer cells. The cell cycle profile was determined by treating AGS cells with the indicated concentration of MLN8237 for 72 h, staining the cells with propidium iodide staining and analyzing by FCM. This staining is representative of three different independent experiments. This immunoblot is representative of three independent experiments. Asterisk (*) indicates a significant difference. *** P

    Article Snippet: To measure apoptosis, mitochondrial membrane potential and Calcium influxes, cells were harvested and stained with FITC Apoptosis Detection Dye, JC-1, Fluo-3 AM (Beyotime Biotechnology), and then analyzed by flow cytometry.

    Techniques: MTS Assay, Staining, Double Staining, Concentration Assay

    MLN8237 treatment activated the calpain pathway and induced apoptosis through p27 degradation and Bax cleavage in AGS cell line. a MLN8237 treatment led to p27 downregulation and Bax cleavage. AGS cells were cultured at the indicated concentration of MLN8237 for 72 h and incubated time at 200 nM. The protein samples were subjected to an immunoblotting analysis of p21, p27, c-Bax, c-PARP, and Actin. b Calpain knockdown attenuated the p27 turnover and Bax cleavage induced by the treatment of the cells with 200 nM MLN8237 for 72 h. Calpain 4 was knocked down and treated with 200 nM MLN8237 for 72 h in AGS. Protein expression was determined by immunoblotting analysis. c Immunoblotting analysis demonstrated that both calpain 1 and calpain 2 degraded p27 and cleaved Bax. Calpain 1/4 or calpain 2/4 were overexpressed in AGS gastric cancer cells and then treated with MLN8237 at 200 nM, and subjected to immunoblotting analysis for indicated protein expression. d Calpain 1 or calpain 2 enhanced apoptosis of MLN8237-treated AGS gastric cancer cells. Calpain 1/4 or calpain 2/4 were overexpressed and treated with 200 nM MLN8237 for 72 h. Apoptosis was determined by FCM. The experiment was performed independently three times. e Both p27 and Bax combined with calpain. Calpain 1/4-Flag was overexpressed with pCMV-p27 or Bax-HA plasmids for 24 h in 293 T. Equivalent cell lysate was immunoprecipitated with Flag beads and immunoblotted p27 and HA protein levels. f , g MLN8237-induced Ca 2+ release to the cytoplasm. AGS was treated with MLN8237 for 72 h at indicated concentration and loaded with 5 μM Fluo-3/AM for 30 min at 37 °C. The labeled cells were analysis by FCM ( f ). g AGS was pre-incubated with 1 and 2 uM EDTA for 2 h and followed treated with 200 nM MLN8237 for 72 h. Error bars represent SD from three independent experiments. Asterisk (*) indicates a significant difference. ** P

    Journal: Cell Death & Disease

    Article Title: Suppression of AURKA alleviates p27 inhibition on Bax cleavage and induces more intensive apoptosis in gastric cancer

    doi: 10.1038/s41419-018-0823-3

    Figure Lengend Snippet: MLN8237 treatment activated the calpain pathway and induced apoptosis through p27 degradation and Bax cleavage in AGS cell line. a MLN8237 treatment led to p27 downregulation and Bax cleavage. AGS cells were cultured at the indicated concentration of MLN8237 for 72 h and incubated time at 200 nM. The protein samples were subjected to an immunoblotting analysis of p21, p27, c-Bax, c-PARP, and Actin. b Calpain knockdown attenuated the p27 turnover and Bax cleavage induced by the treatment of the cells with 200 nM MLN8237 for 72 h. Calpain 4 was knocked down and treated with 200 nM MLN8237 for 72 h in AGS. Protein expression was determined by immunoblotting analysis. c Immunoblotting analysis demonstrated that both calpain 1 and calpain 2 degraded p27 and cleaved Bax. Calpain 1/4 or calpain 2/4 were overexpressed in AGS gastric cancer cells and then treated with MLN8237 at 200 nM, and subjected to immunoblotting analysis for indicated protein expression. d Calpain 1 or calpain 2 enhanced apoptosis of MLN8237-treated AGS gastric cancer cells. Calpain 1/4 or calpain 2/4 were overexpressed and treated with 200 nM MLN8237 for 72 h. Apoptosis was determined by FCM. The experiment was performed independently three times. e Both p27 and Bax combined with calpain. Calpain 1/4-Flag was overexpressed with pCMV-p27 or Bax-HA plasmids for 24 h in 293 T. Equivalent cell lysate was immunoprecipitated with Flag beads and immunoblotted p27 and HA protein levels. f , g MLN8237-induced Ca 2+ release to the cytoplasm. AGS was treated with MLN8237 for 72 h at indicated concentration and loaded with 5 μM Fluo-3/AM for 30 min at 37 °C. The labeled cells were analysis by FCM ( f ). g AGS was pre-incubated with 1 and 2 uM EDTA for 2 h and followed treated with 200 nM MLN8237 for 72 h. Error bars represent SD from three independent experiments. Asterisk (*) indicates a significant difference. ** P

    Article Snippet: To measure apoptosis, mitochondrial membrane potential and Calcium influxes, cells were harvested and stained with FITC Apoptosis Detection Dye, JC-1, Fluo-3 AM (Beyotime Biotechnology), and then analyzed by flow cytometry.

    Techniques: Cell Culture, Concentration Assay, Incubation, Expressing, Immunoprecipitation, Labeling

    c-Bax localized to mitochondria and induced cytochrome c release and led to apoptosis. a Mitochondrial isolation to detect c-Bax distribution and cytochrome c release upon 200 nM MLN8237 treatment for 72 h in AGS. Mitochondrial isolation was conducted according to the manufacturer protocol and measured indicated protein expression in the various component of the cell lysate. b Location detection of EGFP-Bax and EGFP-ΔBax in 293 T. EGFP-Bax or EGFP-ΔBax was transfected into 293 T and photographed through a fluorescence microscope before incubated with mitotracker for 15 min at 37 °C. c Enhanced c-Bax localized to the mitochondrial after p27 silencing in 200 nM MLN8237-treated AGS cell line for 72 h. Cells were subjected to mitochondrial isolation and immunoblotting for quantification of mitochondrial c-Bax. d Augmented deterioration of the mitochondrial membrane potential (MMP) by p27 silencing in MLN8237-treated AGS cell line. AGS cells transfected with negative control or p27 siRNA were treated with 200 nM MLN8237 for 72 h, and subjected to JC-1 staining and FCM to detect MMP collapse. Error bars represent SD from three independent experiments. Representative microscopic images are shown. This immunoblot is representative of three independent experiments. Asterisk (*) indicates a significant difference. ** P

    Journal: Cell Death & Disease

    Article Title: Suppression of AURKA alleviates p27 inhibition on Bax cleavage and induces more intensive apoptosis in gastric cancer

    doi: 10.1038/s41419-018-0823-3

    Figure Lengend Snippet: c-Bax localized to mitochondria and induced cytochrome c release and led to apoptosis. a Mitochondrial isolation to detect c-Bax distribution and cytochrome c release upon 200 nM MLN8237 treatment for 72 h in AGS. Mitochondrial isolation was conducted according to the manufacturer protocol and measured indicated protein expression in the various component of the cell lysate. b Location detection of EGFP-Bax and EGFP-ΔBax in 293 T. EGFP-Bax or EGFP-ΔBax was transfected into 293 T and photographed through a fluorescence microscope before incubated with mitotracker for 15 min at 37 °C. c Enhanced c-Bax localized to the mitochondrial after p27 silencing in 200 nM MLN8237-treated AGS cell line for 72 h. Cells were subjected to mitochondrial isolation and immunoblotting for quantification of mitochondrial c-Bax. d Augmented deterioration of the mitochondrial membrane potential (MMP) by p27 silencing in MLN8237-treated AGS cell line. AGS cells transfected with negative control or p27 siRNA were treated with 200 nM MLN8237 for 72 h, and subjected to JC-1 staining and FCM to detect MMP collapse. Error bars represent SD from three independent experiments. Representative microscopic images are shown. This immunoblot is representative of three independent experiments. Asterisk (*) indicates a significant difference. ** P

    Article Snippet: To measure apoptosis, mitochondrial membrane potential and Calcium influxes, cells were harvested and stained with FITC Apoptosis Detection Dye, JC-1, Fluo-3 AM (Beyotime Biotechnology), and then analyzed by flow cytometry.

    Techniques: Isolation, Expressing, Transfection, Fluorescence, Microscopy, Incubation, Negative Control, Staining

    The protective role of cytoplasmic p27 in MLN8237-induced Bax cleavage and apoptosis in AGS gastric cancer cell line. a p27 distribution in the cytoplasm of AGS. Immunoblotting was subjected to detect p27 distribution after nuclear isolation according to manufacturer description. b p27 and phosphomimetic p27 mutant (T157D or T198D) overexpression inhibited MLN8237-induced Bax cleavage. AGS cells were transfected with Flag-tagged p27 or p27 mutant (T157D or T198D) and then treated with 200 nM MLN8237 for 72 h. c – f The combination of MLN8237 treatment and p27 silencing increased Bax cleavage. AGS cells were subjected to immunoblotting analysis c to determine the expression of the indicated proteins. MTS d was utilized to detect cell proliferation or FCM e , f to detect apoptosis level. 200 nM MLN8237 was incubated for 72 h in AGS. The mean and SDs of the plots were obtained from three wells within three independent MTS and apoptosis detection assays. The apoptosis staining experiment was performed independently three times. g Enhanced cytotoxicity of some chemotherapeutic agents after p27 silencing. 1 μM doxorubicin (Dox), 1 μM Hydroxycamptothecin (HPT), 0.3 μM MLN4924 or 10 μM oxaliplatin (Oxa) was added to culture after p27 silencing and subjected to MTS assay and immunoblotting at 72 h in AGS cell line. Error bars represent SD from three independent experiments. h The cytoplasmic localization of p27 in selected cancer cells. Immunoblotting was subjected to detect p27 distribution after nuclear isolation according manufacturer description. This immunoblot is representative of three independent experiments. Asterisk (*) indicates a significant difference. * P

    Journal: Cell Death & Disease

    Article Title: Suppression of AURKA alleviates p27 inhibition on Bax cleavage and induces more intensive apoptosis in gastric cancer

    doi: 10.1038/s41419-018-0823-3

    Figure Lengend Snippet: The protective role of cytoplasmic p27 in MLN8237-induced Bax cleavage and apoptosis in AGS gastric cancer cell line. a p27 distribution in the cytoplasm of AGS. Immunoblotting was subjected to detect p27 distribution after nuclear isolation according to manufacturer description. b p27 and phosphomimetic p27 mutant (T157D or T198D) overexpression inhibited MLN8237-induced Bax cleavage. AGS cells were transfected with Flag-tagged p27 or p27 mutant (T157D or T198D) and then treated with 200 nM MLN8237 for 72 h. c – f The combination of MLN8237 treatment and p27 silencing increased Bax cleavage. AGS cells were subjected to immunoblotting analysis c to determine the expression of the indicated proteins. MTS d was utilized to detect cell proliferation or FCM e , f to detect apoptosis level. 200 nM MLN8237 was incubated for 72 h in AGS. The mean and SDs of the plots were obtained from three wells within three independent MTS and apoptosis detection assays. The apoptosis staining experiment was performed independently three times. g Enhanced cytotoxicity of some chemotherapeutic agents after p27 silencing. 1 μM doxorubicin (Dox), 1 μM Hydroxycamptothecin (HPT), 0.3 μM MLN4924 or 10 μM oxaliplatin (Oxa) was added to culture after p27 silencing and subjected to MTS assay and immunoblotting at 72 h in AGS cell line. Error bars represent SD from three independent experiments. h The cytoplasmic localization of p27 in selected cancer cells. Immunoblotting was subjected to detect p27 distribution after nuclear isolation according manufacturer description. This immunoblot is representative of three independent experiments. Asterisk (*) indicates a significant difference. * P

    Article Snippet: To measure apoptosis, mitochondrial membrane potential and Calcium influxes, cells were harvested and stained with FITC Apoptosis Detection Dye, JC-1, Fluo-3 AM (Beyotime Biotechnology), and then analyzed by flow cytometry.

    Techniques: Isolation, Mutagenesis, Over Expression, Transfection, Expressing, Incubation, Staining, MTS Assay

    Acitretin preferentially induces apoptosis in HIV-infected cells. ( a ) Percentage of apoptotic cells determined by annexin V staining of GM-HIV-infected or mock-infected CD4+Tcells after 72 h of treatment. The percentage of apoptotic cells was significantly

    Journal: Nature medicine

    Article Title: Stimulating the RIG-I pathway to kill cells in the latent HIV reservoir following viral reactivation

    doi: 10.1038/nm.4124

    Figure Lengend Snippet: Acitretin preferentially induces apoptosis in HIV-infected cells. ( a ) Percentage of apoptotic cells determined by annexin V staining of GM-HIV-infected or mock-infected CD4+Tcells after 72 h of treatment. The percentage of apoptotic cells was significantly

    Article Snippet: After treatment for 24 hours , the cells were stained with Alexa Fluor 647 Annexin V (Life Technologies, Grand Island, NY) to detect apoptosis (100,000 cells per sample) using the EasyCyte6HT-2L flow cytometer (Millipore, Billerica, MA) according to the manufacturer’s instructions.

    Techniques: Infection, Staining

    Short hairpin RNA (shRNA) knockdown of RIG-I expression markedly inhibits acitretin enhancement of apoptosis, induction of IFN-β and CXCL10 production and preferential depletion of HIV-DNA+ cells. CEM T4 cells were transfected with either RIG-I-shRNA

    Journal: Nature medicine

    Article Title: Stimulating the RIG-I pathway to kill cells in the latent HIV reservoir following viral reactivation

    doi: 10.1038/nm.4124

    Figure Lengend Snippet: Short hairpin RNA (shRNA) knockdown of RIG-I expression markedly inhibits acitretin enhancement of apoptosis, induction of IFN-β and CXCL10 production and preferential depletion of HIV-DNA+ cells. CEM T4 cells were transfected with either RIG-I-shRNA

    Article Snippet: After treatment for 24 hours , the cells were stained with Alexa Fluor 647 Annexin V (Life Technologies, Grand Island, NY) to detect apoptosis (100,000 cells per sample) using the EasyCyte6HT-2L flow cytometer (Millipore, Billerica, MA) according to the manufacturer’s instructions.

    Techniques: shRNA, Expressing, Transfection

    Drp1 silencing attenuates CPT-induced mitochondrial fragmentation and apoptosis. a The effects of CPT on mitochondrial fusion/fission proteins expression in OS cells. Representative immunoblot of the protein levels of Drp1, Opa1, Mfn1 and Mfn2. b Comparison of mitochondrial morphology in the control and Drp1 siRNA cells. Images of mitochondria (red) and nucleus (blue) were collected by confocal microscope. c Drp1 silencing reversed the reduction of cell viability in CPT treated 143B cells. Representative dot plots of Annexin V/PI analysis after CPT treatments in the presence or absence of Drp1 siRNA. d CPT decreased ATP production and increased ADP/ATP ratio of both 143B and MG63 cells. The results were expressed as the means ± SD from three independent experiments. * P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Activation of dynamin-related protein 1 - dependent mitochondria fragmentation and suppression of osteosarcoma by cryptotanshinone

    doi: 10.1186/s13046-018-1008-8

    Figure Lengend Snippet: Drp1 silencing attenuates CPT-induced mitochondrial fragmentation and apoptosis. a The effects of CPT on mitochondrial fusion/fission proteins expression in OS cells. Representative immunoblot of the protein levels of Drp1, Opa1, Mfn1 and Mfn2. b Comparison of mitochondrial morphology in the control and Drp1 siRNA cells. Images of mitochondria (red) and nucleus (blue) were collected by confocal microscope. c Drp1 silencing reversed the reduction of cell viability in CPT treated 143B cells. Representative dot plots of Annexin V/PI analysis after CPT treatments in the presence or absence of Drp1 siRNA. d CPT decreased ATP production and increased ADP/ATP ratio of both 143B and MG63 cells. The results were expressed as the means ± SD from three independent experiments. * P

    Article Snippet: Annexin V apoptosis detection kit (55647) was purchased from BD (USA).

    Techniques: Cycling Probe Technology, Expressing, Microscopy

    Drp1 is required for CPT-mediated apoptosis. a Immuno-staining of Bax and Drp1. Confocal microscope scanning photographs indicated the mitochondrial localization of Drp1 and Bax in CPT treated 143B cells. Red: Mito-Tracker Red CMXRos, Green: Bax and Drp1 as indicated. b Expression of Drp1 and Bax in cytosolic and mitochondrial fractions in OS cells were determined by immunoblotting using anti-Drp1 and anti-Bax antibodies. c Interaction between Bax and Drp1 after CPT treatment. Proteins were extracted from CPT-treated OS cells, and then IP was performed with Bax antibody; Co-IP Drp1 and Bax was detected by Western blotting. d After knocking down Drp1 for 24 h, OS cells were treated with CPT 20 μM for indicated time. Bax expression was determined in both cytosolic and mitochondrial fractions using mouse anti-Bax antibody. Hsp60 and α-tubulin antibodies were used as loading controls for mitochondria and cytosol, respectively. e 143B cells were pretreated with Bax inhibitor peptide V5 (BIP-V5) (50 or 200 μM) for 1 h and incubated with 20 μM CPT for 24 h. Annexin V/PI analysis was used to detect apoptotic cells by flow cytometry followed by CPT treatment. The respective cell percentages in early and late apoptosis for different dose treatment are presented in the qantitative analysis. The results were expressed as the means ± SD from three independent experiments. *P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Activation of dynamin-related protein 1 - dependent mitochondria fragmentation and suppression of osteosarcoma by cryptotanshinone

    doi: 10.1186/s13046-018-1008-8

    Figure Lengend Snippet: Drp1 is required for CPT-mediated apoptosis. a Immuno-staining of Bax and Drp1. Confocal microscope scanning photographs indicated the mitochondrial localization of Drp1 and Bax in CPT treated 143B cells. Red: Mito-Tracker Red CMXRos, Green: Bax and Drp1 as indicated. b Expression of Drp1 and Bax in cytosolic and mitochondrial fractions in OS cells were determined by immunoblotting using anti-Drp1 and anti-Bax antibodies. c Interaction between Bax and Drp1 after CPT treatment. Proteins were extracted from CPT-treated OS cells, and then IP was performed with Bax antibody; Co-IP Drp1 and Bax was detected by Western blotting. d After knocking down Drp1 for 24 h, OS cells were treated with CPT 20 μM for indicated time. Bax expression was determined in both cytosolic and mitochondrial fractions using mouse anti-Bax antibody. Hsp60 and α-tubulin antibodies were used as loading controls for mitochondria and cytosol, respectively. e 143B cells were pretreated with Bax inhibitor peptide V5 (BIP-V5) (50 or 200 μM) for 1 h and incubated with 20 μM CPT for 24 h. Annexin V/PI analysis was used to detect apoptotic cells by flow cytometry followed by CPT treatment. The respective cell percentages in early and late apoptosis for different dose treatment are presented in the qantitative analysis. The results were expressed as the means ± SD from three independent experiments. *P

    Article Snippet: Annexin V apoptosis detection kit (55647) was purchased from BD (USA).

    Techniques: Cycling Probe Technology, Immunostaining, Microscopy, Expressing, Co-Immunoprecipitation Assay, Western Blot, Incubation, Flow Cytometry, Cytometry

    Caspase-3, − 8, and − 9 are involvement in CPT-induced OS cell apoptosis. a The expressions of apoptosis-related proteins were measured by western blotting in OS cells following 20 μM CPT treatment for 24 h. b OS cells were pretreated with Z-VAD-FMK (20 μM) for 1 h and incubated with 20 μM CPT for 24 h. Annexin V/PI analysis was used to detect apoptotic cells by flow cytometry followed by CPT treatment. The respective cell percentages in early and late apoptosis for different dose treatment are presented in the quantitative analysis. The results were expressed as the means ± SD from three independent experiments. ** P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Activation of dynamin-related protein 1 - dependent mitochondria fragmentation and suppression of osteosarcoma by cryptotanshinone

    doi: 10.1186/s13046-018-1008-8

    Figure Lengend Snippet: Caspase-3, − 8, and − 9 are involvement in CPT-induced OS cell apoptosis. a The expressions of apoptosis-related proteins were measured by western blotting in OS cells following 20 μM CPT treatment for 24 h. b OS cells were pretreated with Z-VAD-FMK (20 μM) for 1 h and incubated with 20 μM CPT for 24 h. Annexin V/PI analysis was used to detect apoptotic cells by flow cytometry followed by CPT treatment. The respective cell percentages in early and late apoptosis for different dose treatment are presented in the quantitative analysis. The results were expressed as the means ± SD from three independent experiments. ** P

    Article Snippet: Annexin V apoptosis detection kit (55647) was purchased from BD (USA).

    Techniques: Cycling Probe Technology, Western Blot, Incubation, Flow Cytometry, Cytometry

    CPT triggers mitochondrial fragmentation and apoptosis in OS cells. a Representative dot plots of JC-1 aggregates versus JC-1 monomers in response to various CPT treatments of OS cells. b Representative changes in mitochondrial morphology were detected by confocal microscopy at various concentrations of CPT (as indicated). Images of mitochondria (red) and nucleus (blue) were collected by confocal microscope. c Annexin V/PI analysis by flow cytometry followed by CPT treatment (as indicated). The respective cell percentages in early and late apoptosis for different dose treatment are presented in the qantitative analysis. The results were expressed as the means ± SD from three independent experiments. *P

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Activation of dynamin-related protein 1 - dependent mitochondria fragmentation and suppression of osteosarcoma by cryptotanshinone

    doi: 10.1186/s13046-018-1008-8

    Figure Lengend Snippet: CPT triggers mitochondrial fragmentation and apoptosis in OS cells. a Representative dot plots of JC-1 aggregates versus JC-1 monomers in response to various CPT treatments of OS cells. b Representative changes in mitochondrial morphology were detected by confocal microscopy at various concentrations of CPT (as indicated). Images of mitochondria (red) and nucleus (blue) were collected by confocal microscope. c Annexin V/PI analysis by flow cytometry followed by CPT treatment (as indicated). The respective cell percentages in early and late apoptosis for different dose treatment are presented in the qantitative analysis. The results were expressed as the means ± SD from three independent experiments. *P

    Article Snippet: Annexin V apoptosis detection kit (55647) was purchased from BD (USA).

    Techniques: Cycling Probe Technology, Confocal Microscopy, Microscopy, Flow Cytometry, Cytometry

    The C-terminal of VP2 (331–452aa) is essential to promote the degradation of ORAOV1 and cell apoptosis. (A) Schematics represent the genes encoding the full-length VP2 and truncated VP2 molecules (from Δ1 to Δ3). The numbers indicate the amino acid positions in the molecule. (B) Hela cells were transfected with full-length pRK5-flag-vp2 (WT), the indicated truncated vp2 or empty vectors. After 48 h of transfection, total cells were stained with Annexin PE annexin-V and examined by flow cytometry for apoptosis. (C) Cell samples in B were examined by Western Blot. (D) Percentages of the annexin-V positive cells and the ORAOV1 expression levels in (B) . (E) The portion of VP2 from amino acids 331–452 is responsible for ORAOV1 degradation and VP2-induced apoptosis.

    Journal: Frontiers in Microbiology

    Article Title: VP2 of Infectious Bursal Disease Virus Induces Apoptosis via Triggering Oral Cancer Overexpressed 1 (ORAOV1) Protein Degradation

    doi: 10.3389/fmicb.2017.01351

    Figure Lengend Snippet: The C-terminal of VP2 (331–452aa) is essential to promote the degradation of ORAOV1 and cell apoptosis. (A) Schematics represent the genes encoding the full-length VP2 and truncated VP2 molecules (from Δ1 to Δ3). The numbers indicate the amino acid positions in the molecule. (B) Hela cells were transfected with full-length pRK5-flag-vp2 (WT), the indicated truncated vp2 or empty vectors. After 48 h of transfection, total cells were stained with Annexin PE annexin-V and examined by flow cytometry for apoptosis. (C) Cell samples in B were examined by Western Blot. (D) Percentages of the annexin-V positive cells and the ORAOV1 expression levels in (B) . (E) The portion of VP2 from amino acids 331–452 is responsible for ORAOV1 degradation and VP2-induced apoptosis.

    Article Snippet: Caspase-3, Caspase-8 and Caspase-9 colorimetric assay kits were obtained from BioVision (United States), and PE Annexin-V apoptosis detection kit was purchased from BD Pharmingen (United States).

    Techniques: Transfection, Staining, Flow Cytometry, Cytometry, Western Blot, Expressing

    Overexpression of ORAOV1 suppressed VP2-induced apoptosis. (A) Hela cells in 6-well plate were transfected with 0.3 μg of pEGFP-N1-VP2 or pEGFP-N1 as controls, together with 1.5 μg of pRK5-flag-horaov1 or the same amount of pRK5-flag. (B) Cells treated as in (A) were stained with PE annexin-V, and GFP-positive cells were examined with flow cytometry for apoptosis. (C,D) The percentage of apoptotic cells expressing GFP-VP2 or GFP in the presence of overexpressed ORAOV1. Data are representative of three independent experiments. (E) Hela cells were transfected with 300 ng of pEGFP-vp2 together with indicated doses of pRK5-flag-horaov1 or pRK5-flag. The total amount of plasmid DNA were equalized to 2 μg using an irrelevant vector pcDNA-4.0. (F,G) Cells treated as in (E) were stained with PE annexin-V, and GFP-positive cells were examined with flow cytometry for apoptosis. The percentages of VP2 induced apoptotic cells were reduced by the ORAOV1 expression in a dose-dependent manner. Data are representative of three independent experiments. ∗ p

    Journal: Frontiers in Microbiology

    Article Title: VP2 of Infectious Bursal Disease Virus Induces Apoptosis via Triggering Oral Cancer Overexpressed 1 (ORAOV1) Protein Degradation

    doi: 10.3389/fmicb.2017.01351

    Figure Lengend Snippet: Overexpression of ORAOV1 suppressed VP2-induced apoptosis. (A) Hela cells in 6-well plate were transfected with 0.3 μg of pEGFP-N1-VP2 or pEGFP-N1 as controls, together with 1.5 μg of pRK5-flag-horaov1 or the same amount of pRK5-flag. (B) Cells treated as in (A) were stained with PE annexin-V, and GFP-positive cells were examined with flow cytometry for apoptosis. (C,D) The percentage of apoptotic cells expressing GFP-VP2 or GFP in the presence of overexpressed ORAOV1. Data are representative of three independent experiments. (E) Hela cells were transfected with 300 ng of pEGFP-vp2 together with indicated doses of pRK5-flag-horaov1 or pRK5-flag. The total amount of plasmid DNA were equalized to 2 μg using an irrelevant vector pcDNA-4.0. (F,G) Cells treated as in (E) were stained with PE annexin-V, and GFP-positive cells were examined with flow cytometry for apoptosis. The percentages of VP2 induced apoptotic cells were reduced by the ORAOV1 expression in a dose-dependent manner. Data are representative of three independent experiments. ∗ p

    Article Snippet: Caspase-3, Caspase-8 and Caspase-9 colorimetric assay kits were obtained from BioVision (United States), and PE Annexin-V apoptosis detection kit was purchased from BD Pharmingen (United States).

    Techniques: Over Expression, Transfection, Staining, Flow Cytometry, Cytometry, Expressing, Plasmid Preparation

    Knockdown of ORAOV1 induces apoptosis in a dose-dependent manner. (A–D) Effects of different RNAi constructs on endogenous ORAOV1 expression. (A) Hela cells were transfected with siRNA (RNAi#1–3) or controls targeting human ORAOV1. Forty-eight hours after the second transfection, cell lysates were prepared and examined with Western Blot using anti-ORAOV1 antibody. β-actin expression was used as an internal control. (B) The relative levels of human ORAOV1 in ORAOV1 RNAi-treated Hela cells. The density of protein bands in panel A was quantitated by densitometry, and normalized with the density of the β-actin bands. (C) As in (A) , DF-1 cells were transfected with siRNA (RNAi#1-3) or controls targeting chicken ORAOV1. (D) The relative levels of chicken ORAOV1 expression in the RNAi-treated DF-1 cells. (E–H) Knockdown of ORAOV1 by RNAi induces apoptosis in a dose-dependent manner. RNAi construct#2 in (A) or (C) were used to knockdown ORAOV1 in Hela or DF-1 cells. Forty-eight hours after the second transfection, Hela or DF-1 cells were harvested and stained with PE annexin-V, followed by flow cytometry analysis (E,G) . Percentages of the annexin-V positive cells in the indicated groups were calculated, and the significance of the difference between ORAOV1-RNAi and control-RNAi treatments was performed by ANOVA (F,H) . Data are representative of three independent experiments. ∗ p

    Journal: Frontiers in Microbiology

    Article Title: VP2 of Infectious Bursal Disease Virus Induces Apoptosis via Triggering Oral Cancer Overexpressed 1 (ORAOV1) Protein Degradation

    doi: 10.3389/fmicb.2017.01351

    Figure Lengend Snippet: Knockdown of ORAOV1 induces apoptosis in a dose-dependent manner. (A–D) Effects of different RNAi constructs on endogenous ORAOV1 expression. (A) Hela cells were transfected with siRNA (RNAi#1–3) or controls targeting human ORAOV1. Forty-eight hours after the second transfection, cell lysates were prepared and examined with Western Blot using anti-ORAOV1 antibody. β-actin expression was used as an internal control. (B) The relative levels of human ORAOV1 in ORAOV1 RNAi-treated Hela cells. The density of protein bands in panel A was quantitated by densitometry, and normalized with the density of the β-actin bands. (C) As in (A) , DF-1 cells were transfected with siRNA (RNAi#1-3) or controls targeting chicken ORAOV1. (D) The relative levels of chicken ORAOV1 expression in the RNAi-treated DF-1 cells. (E–H) Knockdown of ORAOV1 by RNAi induces apoptosis in a dose-dependent manner. RNAi construct#2 in (A) or (C) were used to knockdown ORAOV1 in Hela or DF-1 cells. Forty-eight hours after the second transfection, Hela or DF-1 cells were harvested and stained with PE annexin-V, followed by flow cytometry analysis (E,G) . Percentages of the annexin-V positive cells in the indicated groups were calculated, and the significance of the difference between ORAOV1-RNAi and control-RNAi treatments was performed by ANOVA (F,H) . Data are representative of three independent experiments. ∗ p

    Article Snippet: Caspase-3, Caspase-8 and Caspase-9 colorimetric assay kits were obtained from BioVision (United States), and PE Annexin-V apoptosis detection kit was purchased from BD Pharmingen (United States).

    Techniques: Construct, Expressing, Transfection, Western Blot, Staining, Flow Cytometry, Cytometry

    Infectious bursal disease virus (IBDV) VP2 induces apoptosis in Hela cells. (A–D) Expression of recombinant GFP-VP2 in Hela cells. Hela cells were transfected with pEGFP-N1-vp2 (A,C) or pEGFP-N1 (B,D) as controls. Twenty-four or 48 h after transfection, cells were stained with DAPI and examined by fluorescence microscope at indicated time points. (E) Transfected cells were collected and stained with PE Annexin-V. GFP-positive cells were gated for further analysis of apoptosis by flow cytometry (total cells were analyzed at 0 h). (F) The percentages of Annexin-V-PE positive cells. (G) The enzymatic activities of caspase-3, -8, and -9 in cells were examined 48 h after transfection with pEGFP-vp2. Data are representative of three independent experiments. ∗∗∗ p

    Journal: Frontiers in Microbiology

    Article Title: VP2 of Infectious Bursal Disease Virus Induces Apoptosis via Triggering Oral Cancer Overexpressed 1 (ORAOV1) Protein Degradation

    doi: 10.3389/fmicb.2017.01351

    Figure Lengend Snippet: Infectious bursal disease virus (IBDV) VP2 induces apoptosis in Hela cells. (A–D) Expression of recombinant GFP-VP2 in Hela cells. Hela cells were transfected with pEGFP-N1-vp2 (A,C) or pEGFP-N1 (B,D) as controls. Twenty-four or 48 h after transfection, cells were stained with DAPI and examined by fluorescence microscope at indicated time points. (E) Transfected cells were collected and stained with PE Annexin-V. GFP-positive cells were gated for further analysis of apoptosis by flow cytometry (total cells were analyzed at 0 h). (F) The percentages of Annexin-V-PE positive cells. (G) The enzymatic activities of caspase-3, -8, and -9 in cells were examined 48 h after transfection with pEGFP-vp2. Data are representative of three independent experiments. ∗∗∗ p

    Article Snippet: Caspase-3, Caspase-8 and Caspase-9 colorimetric assay kits were obtained from BioVision (United States), and PE Annexin-V apoptosis detection kit was purchased from BD Pharmingen (United States).

    Techniques: Expressing, Recombinant, Transfection, Staining, Fluorescence, Microscopy, Flow Cytometry, Cytometry

    ORAOV1 inhibited IBDV induced apoptosis accompanied by restriction of viral release early after infection. (A) DF-1 cells were transfected with pEGFP-horaov1 or pEGFP-N1 as controls. Twenty four hours after transfection, cells were mock infected or infected with IBDV at an MOI of 10. The expressions of GFP-cORAOV1, GFP and actin controls in DF-1 cells were examined with Western Blot using corresponding antibodies. (B) The cell samples in (A) were stained with PE annexin-V, and GFP-positive cells were examined with flow cytometry for apoptosis. (C) Percentages of IBDV-induced apoptotic cells with overexpression of cORAOV1. Data are representative of three independent experiments, and statistically analyzed with ANOVA. ∗∗ p

    Journal: Frontiers in Microbiology

    Article Title: VP2 of Infectious Bursal Disease Virus Induces Apoptosis via Triggering Oral Cancer Overexpressed 1 (ORAOV1) Protein Degradation

    doi: 10.3389/fmicb.2017.01351

    Figure Lengend Snippet: ORAOV1 inhibited IBDV induced apoptosis accompanied by restriction of viral release early after infection. (A) DF-1 cells were transfected with pEGFP-horaov1 or pEGFP-N1 as controls. Twenty four hours after transfection, cells were mock infected or infected with IBDV at an MOI of 10. The expressions of GFP-cORAOV1, GFP and actin controls in DF-1 cells were examined with Western Blot using corresponding antibodies. (B) The cell samples in (A) were stained with PE annexin-V, and GFP-positive cells were examined with flow cytometry for apoptosis. (C) Percentages of IBDV-induced apoptotic cells with overexpression of cORAOV1. Data are representative of three independent experiments, and statistically analyzed with ANOVA. ∗∗ p

    Article Snippet: Caspase-3, Caspase-8 and Caspase-9 colorimetric assay kits were obtained from BioVision (United States), and PE Annexin-V apoptosis detection kit was purchased from BD Pharmingen (United States).

    Techniques: Infection, Transfection, Western Blot, Staining, Flow Cytometry, Cytometry, Over Expression

    Effect of miR-200b-3p on the proliferation and apoptosis of CRC cells. (A) CFSE assay for cell proliferation. (B) Flow cytometric analysis for apoptosis. **P

    Journal: Molecular Medicine Reports

    Article Title: miR-200b-3p inhibits proliferation and induces apoptosis in colorectal cancer by targeting Wnt1

    doi: 10.3892/mmr.2018.9287

    Figure Lengend Snippet: Effect of miR-200b-3p on the proliferation and apoptosis of CRC cells. (A) CFSE assay for cell proliferation. (B) Flow cytometric analysis for apoptosis. **P

    Article Snippet: Flow cytometric analysis of apoptosis The apoptosis of CRC cells was measured with the Annexin V-fluorescein isothiocycanate (FITC)/propidium iodide (PI) apoptosis detection kit (BioVision, Inc., Milpitas, CA, USA).

    Techniques: CFSE Assay, Flow Cytometry

    p53 expression affects the apoptosis rate of hemangioma-derived endothelial cells treated with propranolol (100 µmol/l). *P

    Journal: Molecular Medicine Reports

    Article Title: Propranolol induces hemangioma endothelial cell apoptosis via a p53-BAX mediated pathway

    doi: 10.3892/mmr.2018.9013

    Figure Lengend Snippet: p53 expression affects the apoptosis rate of hemangioma-derived endothelial cells treated with propranolol (100 µmol/l). *P

    Article Snippet: The membranes were blocked with 5% Bovine Serum Albumin (HyClone; GE Healthcare Life Sciences, Logen, UT, USA) at 4°C for 24 h. The membranes were incubated overnight at 4°C with the following primary antibodies: Anti-β-actin (cat. no. ab8226; dilution, 1 mg/ml, molecular weight 45 kDa), antibody against tumor necrosis factor receptor superfamily member 6 (FAS; cat. no. ab133619, dilution 1:1,000), p53-induced death domain-containing protein (PIDD; cat. no. ab78389, dilution 1 µg/ml), death receptor 5 (DR5; cat. no. ab199357, dilution 1:1,000), apoptosis regulator BAX (BAX; cat. no. ab53154, dilution 1:1,000), BH3-interacting domain death agonist (BID; cat. no. ab32060, dilution 1:1,000), p53 unregulated modulator of apoptosis (PUMA; cat. no. ab33906, dilution 1:1,000), insulin-like growth factor-binding protein 3 (IGF-BP3; cat. no. ab77635, dilution 0.03 µg/ml) and phosphatidylinositol-glycan biosynthesis class S protein (PIG-S; cat. no. ab157211, dilution 1:1,000; all Abcam).

    Techniques: Expressing, Derivative Assay

    Signaling pathway of propranolol inducing apoptosis in HemECs in vitro ).

    Journal: Molecular Medicine Reports

    Article Title: Propranolol induces hemangioma endothelial cell apoptosis via a p53-BAX mediated pathway

    doi: 10.3892/mmr.2018.9013

    Figure Lengend Snippet: Signaling pathway of propranolol inducing apoptosis in HemECs in vitro ).

    Article Snippet: The membranes were blocked with 5% Bovine Serum Albumin (HyClone; GE Healthcare Life Sciences, Logen, UT, USA) at 4°C for 24 h. The membranes were incubated overnight at 4°C with the following primary antibodies: Anti-β-actin (cat. no. ab8226; dilution, 1 mg/ml, molecular weight 45 kDa), antibody against tumor necrosis factor receptor superfamily member 6 (FAS; cat. no. ab133619, dilution 1:1,000), p53-induced death domain-containing protein (PIDD; cat. no. ab78389, dilution 1 µg/ml), death receptor 5 (DR5; cat. no. ab199357, dilution 1:1,000), apoptosis regulator BAX (BAX; cat. no. ab53154, dilution 1:1,000), BH3-interacting domain death agonist (BID; cat. no. ab32060, dilution 1:1,000), p53 unregulated modulator of apoptosis (PUMA; cat. no. ab33906, dilution 1:1,000), insulin-like growth factor-binding protein 3 (IGF-BP3; cat. no. ab77635, dilution 0.03 µg/ml) and phosphatidylinositol-glycan biosynthesis class S protein (PIG-S; cat. no. ab157211, dilution 1:1,000; all Abcam).

    Techniques: In Vitro

    Relative expression of different apoptosis related proteins with control and propranolol. Bax, Bcl-2-associated X protein; Bid, BH3 interacting-domain death agonist; Dr5, death receptor 5; Fas, tumor necrosis factor receptor superfamily member 6; Igf-bp3, insulin-like growth factor-binding protein 3; Pidd, p53-induced protein with a death domain; pigs, phosphatidylinositol glycan anchor biosynthesis class S; puma, p53 unregulated modulator of apoptosis.

    Journal: Molecular Medicine Reports

    Article Title: Propranolol induces hemangioma endothelial cell apoptosis via a p53-BAX mediated pathway

    doi: 10.3892/mmr.2018.9013

    Figure Lengend Snippet: Relative expression of different apoptosis related proteins with control and propranolol. Bax, Bcl-2-associated X protein; Bid, BH3 interacting-domain death agonist; Dr5, death receptor 5; Fas, tumor necrosis factor receptor superfamily member 6; Igf-bp3, insulin-like growth factor-binding protein 3; Pidd, p53-induced protein with a death domain; pigs, phosphatidylinositol glycan anchor biosynthesis class S; puma, p53 unregulated modulator of apoptosis.

    Article Snippet: The membranes were blocked with 5% Bovine Serum Albumin (HyClone; GE Healthcare Life Sciences, Logen, UT, USA) at 4°C for 24 h. The membranes were incubated overnight at 4°C with the following primary antibodies: Anti-β-actin (cat. no. ab8226; dilution, 1 mg/ml, molecular weight 45 kDa), antibody against tumor necrosis factor receptor superfamily member 6 (FAS; cat. no. ab133619, dilution 1:1,000), p53-induced death domain-containing protein (PIDD; cat. no. ab78389, dilution 1 µg/ml), death receptor 5 (DR5; cat. no. ab199357, dilution 1:1,000), apoptosis regulator BAX (BAX; cat. no. ab53154, dilution 1:1,000), BH3-interacting domain death agonist (BID; cat. no. ab32060, dilution 1:1,000), p53 unregulated modulator of apoptosis (PUMA; cat. no. ab33906, dilution 1:1,000), insulin-like growth factor-binding protein 3 (IGF-BP3; cat. no. ab77635, dilution 0.03 µg/ml) and phosphatidylinositol-glycan biosynthesis class S protein (PIG-S; cat. no. ab157211, dilution 1:1,000; all Abcam).

    Techniques: Expressing, Binding Assay

    Characterization of apoptotic signaling efficiency of truncated variants of Gal-3 (A) Schematic details of Gal-3 and its variants. (B) SDS-PAGE analysis of Gal-3 and its variants. (C-D) Jurkat cells were treated with 2.5 μM Gal-3 or its variants for 18 h and (C) cellular apoptosis was assessed by flow cytometry and (D) signaling was assessed by western blot analysis of p-ERK1/2 and cleaved caspase-3.

    Journal: Oncotarget

    Article Title: The N-terminal tail coordinates with carbohydrate recognition domain to mediate galectin-3 induced apoptosis in T cells

    doi: 10.18632/oncotarget.17760

    Figure Lengend Snippet: Characterization of apoptotic signaling efficiency of truncated variants of Gal-3 (A) Schematic details of Gal-3 and its variants. (B) SDS-PAGE analysis of Gal-3 and its variants. (C-D) Jurkat cells were treated with 2.5 μM Gal-3 or its variants for 18 h and (C) cellular apoptosis was assessed by flow cytometry and (D) signaling was assessed by western blot analysis of p-ERK1/2 and cleaved caspase-3.

    Article Snippet: Flow cytometry analysis To analyze apoptosis, cells were harvested after treatments, washed thrice with PBS, re-suspended in the binding buffer (PI/FITC-Annexin V Apoptosis Detection Kit (KGA 107) from KeyGEN Biotech (Nanjing, China)), and stained with PI/FITC-AnnexinV for 30 min according to the manufacturer's instructions.

    Techniques: SDS Page, Flow Cytometry, Cytometry, Western Blot

    Roles of T cell surface receptor molecules in Gal-3-triggered T cell apoptosis (A) Jurkat cells were transfected with siRNAs to CD45, CD29 or CD71 and compared with the negative control siRNA (siNC). The knockdown efficiency of CD45, CD29 and CD71 was determined by western blotting. (B) Jurkat cells from (A) were incubated with or without 2.5 μM Gal-3 (c refers to control). P-ERK1/2 and cleaved caspase-3 was determined by western blotting. (C-D) Control or CD45 knocked-down Jurkat cells were incubated with or without 2.5 μM Gal-3 and p-PKC was determined by western blotting (C) and ROS production was analyzed by flow cytometry (D). (E) Control or CD45 knocked-down Jurkat cells were incubated with or without 2.5 μM G69-250 variant and then analyzed for CD45, p-ERK, p-PKC and cleaved caspase-3. (F) Jurkat cell lysate was incubated with His-Gal-3- or His-G69-250-immobilized Ni-NTA beads. The upper panel shows CD45 determined by western blotting and the lower panel shows SDS-PAGE analysis of His-Gal-3 or His-G69-250 on the beads.

    Journal: Oncotarget

    Article Title: The N-terminal tail coordinates with carbohydrate recognition domain to mediate galectin-3 induced apoptosis in T cells

    doi: 10.18632/oncotarget.17760

    Figure Lengend Snippet: Roles of T cell surface receptor molecules in Gal-3-triggered T cell apoptosis (A) Jurkat cells were transfected with siRNAs to CD45, CD29 or CD71 and compared with the negative control siRNA (siNC). The knockdown efficiency of CD45, CD29 and CD71 was determined by western blotting. (B) Jurkat cells from (A) were incubated with or without 2.5 μM Gal-3 (c refers to control). P-ERK1/2 and cleaved caspase-3 was determined by western blotting. (C-D) Control or CD45 knocked-down Jurkat cells were incubated with or without 2.5 μM Gal-3 and p-PKC was determined by western blotting (C) and ROS production was analyzed by flow cytometry (D). (E) Control or CD45 knocked-down Jurkat cells were incubated with or without 2.5 μM G69-250 variant and then analyzed for CD45, p-ERK, p-PKC and cleaved caspase-3. (F) Jurkat cell lysate was incubated with His-Gal-3- or His-G69-250-immobilized Ni-NTA beads. The upper panel shows CD45 determined by western blotting and the lower panel shows SDS-PAGE analysis of His-Gal-3 or His-G69-250 on the beads.

    Article Snippet: Flow cytometry analysis To analyze apoptosis, cells were harvested after treatments, washed thrice with PBS, re-suspended in the binding buffer (PI/FITC-Annexin V Apoptosis Detection Kit (KGA 107) from KeyGEN Biotech (Nanjing, China)), and stained with PI/FITC-AnnexinV for 30 min according to the manufacturer's instructions.

    Techniques: Cell Surface Receptor Assay, Transfection, Negative Control, Western Blot, Incubation, Flow Cytometry, Cytometry, Variant Assay, SDS Page

    Gal-3-induced T cell apoptosis is inhibited by NT or CRD inhibitors Jurkat cells were incubated with 1 μM Gal-3 for 18 h in the presence or absence of (A) 10 or 15 μM G1-108 variant, (B) 10 or 20 μg/ml A3A12 antibody or (C) 10 or 15 μM G111-250 variant. Caspase-3 cleavage was assessed by western blot. (D-E) Jurkat cells were treated with 2.5 μM Gal-3 in the absence or presence of 0.1, 0.5 and 2 mg/ml MCP for 18 h. The apoptotic rate was measured by flow cytometry (D), and p-ERK1/2 and cleaved caspase-3 were determined by western blotting (E). The data are representative of three independent experiments.

    Journal: Oncotarget

    Article Title: The N-terminal tail coordinates with carbohydrate recognition domain to mediate galectin-3 induced apoptosis in T cells

    doi: 10.18632/oncotarget.17760

    Figure Lengend Snippet: Gal-3-induced T cell apoptosis is inhibited by NT or CRD inhibitors Jurkat cells were incubated with 1 μM Gal-3 for 18 h in the presence or absence of (A) 10 or 15 μM G1-108 variant, (B) 10 or 20 μg/ml A3A12 antibody or (C) 10 or 15 μM G111-250 variant. Caspase-3 cleavage was assessed by western blot. (D-E) Jurkat cells were treated with 2.5 μM Gal-3 in the absence or presence of 0.1, 0.5 and 2 mg/ml MCP for 18 h. The apoptotic rate was measured by flow cytometry (D), and p-ERK1/2 and cleaved caspase-3 were determined by western blotting (E). The data are representative of three independent experiments.

    Article Snippet: Flow cytometry analysis To analyze apoptosis, cells were harvested after treatments, washed thrice with PBS, re-suspended in the binding buffer (PI/FITC-Annexin V Apoptosis Detection Kit (KGA 107) from KeyGEN Biotech (Nanjing, China)), and stained with PI/FITC-AnnexinV for 30 min according to the manufacturer's instructions.

    Techniques: Incubation, Variant Assay, Western Blot, Flow Cytometry, Cytometry

    Model for Gal-3-induced T cell apoptosis The interaction of Gal-3 with cell surface death receptors activates ERK via ROS and PKC. The death signal is then conveyed from ERK to caspase-9 that activates caspase-3 and the downstream pathways that ultimately result in cell apoptosis. As with full length Gal-3, the truncated G13-250 variant can activate both pathways, whereas the G69-250 variant can only activate the PKC pathway. The G111-250 and G1-108 variants were unable to activate both pathways.

    Journal: Oncotarget

    Article Title: The N-terminal tail coordinates with carbohydrate recognition domain to mediate galectin-3 induced apoptosis in T cells

    doi: 10.18632/oncotarget.17760

    Figure Lengend Snippet: Model for Gal-3-induced T cell apoptosis The interaction of Gal-3 with cell surface death receptors activates ERK via ROS and PKC. The death signal is then conveyed from ERK to caspase-9 that activates caspase-3 and the downstream pathways that ultimately result in cell apoptosis. As with full length Gal-3, the truncated G13-250 variant can activate both pathways, whereas the G69-250 variant can only activate the PKC pathway. The G111-250 and G1-108 variants were unable to activate both pathways.

    Article Snippet: Flow cytometry analysis To analyze apoptosis, cells were harvested after treatments, washed thrice with PBS, re-suspended in the binding buffer (PI/FITC-Annexin V Apoptosis Detection Kit (KGA 107) from KeyGEN Biotech (Nanjing, China)), and stained with PI/FITC-AnnexinV for 30 min according to the manufacturer's instructions.

    Techniques: Variant Assay

    ERK phosphorylation is induced by Gal-3 in Jurkat cells (A) Jurkat cells were incubated with Gal-3 in the presence of U0126 or lactose for 10 min, 1 h, 6 h or 18 h and the apoptosis was measured by flow cytometry after PI/FITC-Annexin V double staining or (B) western blotting. (C) Jurkat cells were incubated with Gal-3 for 10 min, 1 h, 6 h or 18 h in the presence of U0126, which was added 1 h before (−1h) or 10 min, 1 h or 6 h after addition of Gal-3. P-ERK1/2 was then assessed by western blotting. (D) Jurkat cells were incubated with Gal-3 for 18 h in the presence of U0126 as described in C and the cleavage of caspase-3 was determined by western blotting. (E) Jurkat cells were treated with Gal-3 for 18 h in the presence or absence of U0126 and the cleavage of caspase-9 and caspase-3 were analyzed by western blotting. The data are representative of three independent experiments.

    Journal: Oncotarget

    Article Title: The N-terminal tail coordinates with carbohydrate recognition domain to mediate galectin-3 induced apoptosis in T cells

    doi: 10.18632/oncotarget.17760

    Figure Lengend Snippet: ERK phosphorylation is induced by Gal-3 in Jurkat cells (A) Jurkat cells were incubated with Gal-3 in the presence of U0126 or lactose for 10 min, 1 h, 6 h or 18 h and the apoptosis was measured by flow cytometry after PI/FITC-Annexin V double staining or (B) western blotting. (C) Jurkat cells were incubated with Gal-3 for 10 min, 1 h, 6 h or 18 h in the presence of U0126, which was added 1 h before (−1h) or 10 min, 1 h or 6 h after addition of Gal-3. P-ERK1/2 was then assessed by western blotting. (D) Jurkat cells were incubated with Gal-3 for 18 h in the presence of U0126 as described in C and the cleavage of caspase-3 was determined by western blotting. (E) Jurkat cells were treated with Gal-3 for 18 h in the presence or absence of U0126 and the cleavage of caspase-9 and caspase-3 were analyzed by western blotting. The data are representative of three independent experiments.

    Article Snippet: Flow cytometry analysis To analyze apoptosis, cells were harvested after treatments, washed thrice with PBS, re-suspended in the binding buffer (PI/FITC-Annexin V Apoptosis Detection Kit (KGA 107) from KeyGEN Biotech (Nanjing, China)), and stained with PI/FITC-AnnexinV for 30 min according to the manufacturer's instructions.

    Techniques: Incubation, Flow Cytometry, Cytometry, Double Staining, Western Blot

    ROS and PKC activation is necessary for Gal-3-induced apoptosis (A) Jurkat cells were incubated with Gal-3 for 10 min, 1 h, 6 h and 18 h in the absence or presence of NAC, U0126, AEB071, or lactose and analyzed by flow cytometry for ROS as described in the Materials and Methods. (B) Jurkat cells were treated with Gal-3 for 10 min, 1 h, 6 h and 18 h and analyzed for p-PKC by western blotting. (C) Jurkat cells were incubated with Gal-3 or PMA in the absence or presence of AEB071 for 18 h and analyzed for p-PKC by western blotting. (D) Jurkat cells were treated with Gal-3 for 18 h in the absence or presence of AEB071 and NAC and were analyzed for p-ERK and cleaved caspase-3. (E) Jurkat cells were incubated with Gal-3 in the absence or presence of NAC for 18 h. p-PKC was analyzed by western blotting. The data are representative of three independent experiments.

    Journal: Oncotarget

    Article Title: The N-terminal tail coordinates with carbohydrate recognition domain to mediate galectin-3 induced apoptosis in T cells

    doi: 10.18632/oncotarget.17760

    Figure Lengend Snippet: ROS and PKC activation is necessary for Gal-3-induced apoptosis (A) Jurkat cells were incubated with Gal-3 for 10 min, 1 h, 6 h and 18 h in the absence or presence of NAC, U0126, AEB071, or lactose and analyzed by flow cytometry for ROS as described in the Materials and Methods. (B) Jurkat cells were treated with Gal-3 for 10 min, 1 h, 6 h and 18 h and analyzed for p-PKC by western blotting. (C) Jurkat cells were incubated with Gal-3 or PMA in the absence or presence of AEB071 for 18 h and analyzed for p-PKC by western blotting. (D) Jurkat cells were treated with Gal-3 for 18 h in the absence or presence of AEB071 and NAC and were analyzed for p-ERK and cleaved caspase-3. (E) Jurkat cells were incubated with Gal-3 in the absence or presence of NAC for 18 h. p-PKC was analyzed by western blotting. The data are representative of three independent experiments.

    Article Snippet: Flow cytometry analysis To analyze apoptosis, cells were harvested after treatments, washed thrice with PBS, re-suspended in the binding buffer (PI/FITC-Annexin V Apoptosis Detection Kit (KGA 107) from KeyGEN Biotech (Nanjing, China)), and stained with PI/FITC-AnnexinV for 30 min according to the manufacturer's instructions.

    Techniques: Activation Assay, Incubation, Flow Cytometry, Cytometry, Western Blot

    Gal-3 treatment induces Jurkat cell apoptosis (A) Jurkat cells were incubated with 2.5 μM Gal-3 for 10 min, 1 h, 6 h and 18 h and apoptosis was analyzed by PI/FITC-AnnexinV double staining and flow cytometry. (B) Gal-3-treated Jurkat cells were analyzed for the presence of phosphorylated and non-phosphorylated forms of ERK1/2, JNK and p38 MAPKs by western blotting. Also, full length (pro-Casp-3) and cleaved caspase-3 (Cl-Casp-3) were analyzed by western blotting.

    Journal: Oncotarget

    Article Title: The N-terminal tail coordinates with carbohydrate recognition domain to mediate galectin-3 induced apoptosis in T cells

    doi: 10.18632/oncotarget.17760

    Figure Lengend Snippet: Gal-3 treatment induces Jurkat cell apoptosis (A) Jurkat cells were incubated with 2.5 μM Gal-3 for 10 min, 1 h, 6 h and 18 h and apoptosis was analyzed by PI/FITC-AnnexinV double staining and flow cytometry. (B) Gal-3-treated Jurkat cells were analyzed for the presence of phosphorylated and non-phosphorylated forms of ERK1/2, JNK and p38 MAPKs by western blotting. Also, full length (pro-Casp-3) and cleaved caspase-3 (Cl-Casp-3) were analyzed by western blotting.

    Article Snippet: Flow cytometry analysis To analyze apoptosis, cells were harvested after treatments, washed thrice with PBS, re-suspended in the binding buffer (PI/FITC-Annexin V Apoptosis Detection Kit (KGA 107) from KeyGEN Biotech (Nanjing, China)), and stained with PI/FITC-AnnexinV for 30 min according to the manufacturer's instructions.

    Techniques: Incubation, Double Staining, Flow Cytometry, Cytometry, Western Blot