Journal: Disease Models & Mechanisms

Article Title: TNF receptors regulate vascular homeostasis in zebrafish through a caspase-8, caspase-2 and P53 apoptotic program that bypasses caspase-3

doi: 10.1242/dmm.010249

Figure Lengend Snippet: TNFRSF1B depletion promotes a caspase-8 apoptotic pathway via TNFRSF1A in endothelial cells. (A-D) Wild-type and P53 M214K zebrafish embryos were microinjected at the one-cell stage with standard, TNFRSF1A, TNFRSF1B, P53, CASP8 and CASP9 MOs alone or in combination with the mRNAs encoding the caspase-8 inhibitors CFLAR and CMRA. (A) The activation of caspase-8 and caspase-9 in whole embryos was quantified at 60 hpf using the luminescent assays Caspase-Glo 8 and Caspase-Glo 9. Represented are mean values + s.e.m. (B-D) Vascular defects scored at 72 hpf as indicated in the legend to . Different letters denote statistically significant differences among the groups according to a Tukey test. Note that inhibition of the caspase-8 but not caspase-9 pathway partially rescues the TNFRSF1B vascular phenotype. * P <0.05, ** P <0.01, *** P <0.0001; n.s., not significant according to a Chi-square contingency test (B-D).

Article Snippet: After blocking, embryos were incubated overnight at 4°C with 0.5 μg/ml affinity-purified rabbit anti-human/mouse caspase-3 active or anti-zebrafish P53 (1:200; #55915, AnaSpec) diluted in PDT, washed six times in PDT (10 minutes each), incubated for 2 hours at room temperature in PDT, incubated overnight at 4°C with a 1:1000 dilution in PDT of a phycoerythrin-conjugated secondary antibody (Invitrogen), washed five times in PBT (5 minutes each) and finally examined under a LEICA MZ16FA stereomicroscope.

Techniques: Activation Assay, Inhibition

Journal: Disease Models & Mechanisms

Article Title: TNF receptors regulate vascular homeostasis in zebrafish through a caspase-8, caspase-2 and P53 apoptotic program that bypasses caspase-3

doi: 10.1242/dmm.010249

Figure Lengend Snippet: P53 activation is indispensable for promoting caspase-8-dependent apoptosis of endothelial cell in TNFRSF1B-deficient zebrafish. (A-H) Wild-type (A,B,D-G), transgenic fli1a :eGFP (C) and P53 M214K (H) zebrafish embryos were microinjected at the one-cell stage with standard, TNFRSF1 A, TNFRSF1B and P53 MOs. At 72 hpf, the mRNA levels of the indicated genes were determined by real-time RT-PCR in 10 pooled larvae (A,D,E,G,H). The gene expression is normalized against rps11, each bar represents the mean + s.e.m. Different letters denote statistically significant differences among the groups according to a Tukey test. * P <0.05; n.s., not significant according to Chi-square contingency (F) or Student's t tests (D,E,H). (A) Co-injection of TNFRSF 1A or CASP8 MOs reduces TNFRSF1B-MO-mediated P53 upregulation. (B) P53 protein levels were assayed by western blot in dechorionated and deyolked embryos at 24 hpf. Note that P53 levels are upregulated in TNFRSF 1B-deficient larvae. (C) Whole mount immunohistochemistry against P53 at 3 dpf in Tg( fil1a :eGFP) larvae injected with standard or TNFRSF1B MOs. Note a massive increase in P53 expression (red) in the vascular endothelium (green) of TNFRSF1B-deficient larvae. (D,E) TNFRSF1B silencing leads to upregulation of box , mdm2 and p21. (F) Vascular defects in larvae deficient in both TNFRSF 1B and P53 were scored as indicated in the legend to . Note that P53 deficiency partially rescues the vascular defects observed in TNFRSF 1B-deficient fish. (G) Upregulation of caspase-8 by TNFRSF 1B deficiency is dependent on TNFRSF 1A and P53. (H) p21 mRNA levels in P53 mutant larvae are not altered by TNFRSF 1B silencing. (I) Representation of the proposed amplification loop between extrinsic and intrinsic apoptotic pathways triggered by TNFRSF 1A. CA, caudal artery; CV, caudal vein; ISV, intersegmental vessels. Scale bars: 25 μm.

Article Snippet: After blocking, embryos were incubated overnight at 4°C with 0.5 μg/ml affinity-purified rabbit anti-human/mouse caspase-3 active or anti-zebrafish P53 (1:200; #55915, AnaSpec) diluted in PDT, washed six times in PDT (10 minutes each), incubated for 2 hours at room temperature in PDT, incubated overnight at 4°C with a 1:1000 dilution in PDT of a phycoerythrin-conjugated secondary antibody (Invitrogen), washed five times in PBT (5 minutes each) and finally examined under a LEICA MZ16FA stereomicroscope.

Techniques: Activation Assay, Transgenic Assay, Quantitative RT-PCR, Gene Expression, Injection, Western Blot, Immunohistochemistry, Expressing, Mutagenesis, Amplification

Journal: Disease Models & Mechanisms

Article Title: TNF receptors regulate vascular homeostasis in zebrafish through a caspase-8, caspase-2 and P53 apoptotic program that bypasses caspase-3

doi: 10.1242/dmm.010249

Figure Lengend Snippet: DNA fragmentation in endothelial cells triggers P53 activation in TNFRSF1B-deficient zebrafish. (A,B) Wild-type zebrafish embryos were microinjected at the one-cell stage with standard and/or TNFRSF1B MOs alone or in combination with the mRNAs encoding ICAD or the antisense mRNA for TNFRSF 1B, as an appropriate control. Some larvae were treated at 48 hpf by bath immersion for 16 hours with 1 μM of the CHK1 specific inhibitor Gö6976. Vascular defects were scored at 72 hpf as indicated in the legend to . * P <0.05, ** P <0.01, *** P <0.0001; n.s., not significant according to a Chi-square contingency test.

Article Snippet: After blocking, embryos were incubated overnight at 4°C with 0.5 μg/ml affinity-purified rabbit anti-human/mouse caspase-3 active or anti-zebrafish P53 (1:200; #55915, AnaSpec) diluted in PDT, washed six times in PDT (10 minutes each), incubated for 2 hours at room temperature in PDT, incubated overnight at 4°C with a 1:1000 dilution in PDT of a phycoerythrin-conjugated secondary antibody (Invitrogen), washed five times in PBT (5 minutes each) and finally examined under a LEICA MZ16FA stereomicroscope.

Techniques: Activation Assay, Control

Journal: Disease Models & Mechanisms

Article Title: TNF receptors regulate vascular homeostasis in zebrafish through a caspase-8, caspase-2 and P53 apoptotic program that bypasses caspase-3

doi: 10.1242/dmm.010249

Figure Lengend Snippet: The caspase-2–P53 apoptotic signaling pathway triggered by TNFRSF1A is evolutionarily conserved. (A-F) HUVEC were treated for 16 hours with 40 ng/ml human TNFα (hTNFα) alone or in combination with 5 μg/ml of a mouse IgG isotype control or a neutralizing antibody to human TNFRSF1B (αhTNFRSF1B) (A,E) or with a TNFα mutein specific for TNFRSF1A (B-D,F). (A) Cell viability was determined using the MTT colorimetric assay. (B,C) PS flip and caspase-2 activity were evaluated by flow cytometry and immunofluorescence using phycoerythrin-Annexin V conjugate and the CaspGLOW Fluorescein Active Caspase-2 Staining kit, respectively. Note that Annexin V + cells showed membrane blebbing (C, arrowheads). BF, bright field. (D) Caspase-3 activation was determined by immunofluorescence using a specific antibody to active caspase-3. Some cultures were treated for 16 hours with 0.5 μM camptothecin (campt), 1 μM Gö6976 or 0.5 μM staurosporine in the presence or absence of 50 μM of the caspase-3 inhibitor Ac-DEVD-CHO. (E) mRNA levels of the indicated genes were determined by real-time RT-PCR, normalized against rps11, and shown relative to non-treated cells (mean + s.e.m.). (F) P53 protein levels were assayed by western blot using monoclonal antibodies to human P53 (TP53) and β-actin (ACTB). Different letters denote statistically significant differences between the groups according to a Tukey test. The groups labeled with ‘a’ or ‘A’ in E were not statistically significant from non-treated cells. * P <0.05 according to a Student's t -test; ND, not determined.

Article Snippet: After blocking, embryos were incubated overnight at 4°C with 0.5 μg/ml affinity-purified rabbit anti-human/mouse caspase-3 active or anti-zebrafish P53 (1:200; #55915, AnaSpec) diluted in PDT, washed six times in PDT (10 minutes each), incubated for 2 hours at room temperature in PDT, incubated overnight at 4°C with a 1:1000 dilution in PDT of a phycoerythrin-conjugated secondary antibody (Invitrogen), washed five times in PBT (5 minutes each) and finally examined under a LEICA MZ16FA stereomicroscope.

Techniques: Control, Colorimetric Assay, Activity Assay, Flow Cytometry, Immunofluorescence, Staining, Membrane, Activation Assay, Quantitative RT-PCR, Western Blot, Bioprocessing, Labeling