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

Journal: Virologica Sinica

Article Title: Cutaneous Dengue Virus Inoculation Triggers Strong B Cell Reactions but Contrastingly Poor T Cell Responses

doi: 10.1007/s12250-020-00213-6

Figure Lengend Snippet: T cells do not seem to undergo significant apoptosis after DENV inoculation. Regional DLNs were frozen after the corresponding treatments. A DLN cryosections were stained with H&E. Black arrows in the images as well as the magnifications below indicate apoptotic bodies. Scale bars 50 µm. B Immunofluorescence in tissue sections to detect active caspase-3 (green) in the paracortical area (Thy 1.2+, red). Nuclei were stained with DAPI (blue). Magnification below show caspase 3+ T cells in DENV-infected tissue. C White arrows indicate T cells (Thy 1.2+, red fluorescence) with fragmented DNA (TUNEL+, green fluorescence) in situ . Scale bars 50 µm. Floating bar charts show the mean ± SEM of the numbers of apoptotic cells in the T cell zone (TCZ) after DENV infection and those in the PBS-control DLNs. Data are representative of two independent experiments from 4 mice each. Statistical significance was calculated using unpaired two-tailed Student’s t -test. ns not significantly different.

Article Snippet: Some slides were stain with Hematoxilin and Eosin (H&E) following conventional histological protocols and others were rehydrated in PBS-0.01% Tween-20, blocked with a casein solution (Power Block, BioGenex Laboratories, San Ramon, CA, USA) and labeled with the following primary antibodies in a PBS solution containing 1% (vol/vol) of bovine serum albumin, 1% (vol/vol) of normal human serum and 0.01% of sodium azide: Rat anti-mouse B220-Brilliant Violet 450 from BioLegend (RA3-6B2; San Diego, CA, USA), Rat anti-mouse Thy 1.2-Biotin (53-2.1) and Rabbit anti-mouse Active Caspase-3-FITC (C92-605.1) from BD Biosciences (San Jose, CA, USA), Rabbit anti-mouse Ki-67 (polyclonal) from Abcam (Cambridge, UK), Rat anti-mouse CD68 antibody (FA-11) from BioRad (Hercules, CA, USA) and Sheep anti-mouse IgD antiserum.

Techniques: Staining, Immunofluorescence, Infection, Fluorescence, TUNEL Assay, In Situ, Two Tailed Test

Journal: Pharmaceuticals

Article Title: Mitochondrion-Targeted NIR Therapeutic Agent Suppresses Melanoma by Inducing Apoptosis and Cell Cycle Arrest via E2F/Cyclin/CDK Pathway

doi: 10.3390/ph15121589

Figure Lengend Snippet: IR-817 induced melanoma cell cycle arrest at the G0/G1 phase by E2F/Cyclin/CDK signal regulation network. ( A ) The mRNA expression of E2f8, Ccna2, Rrm2, Cdk1, Cdc45, Ccne1, and E2f7 were validated by qRT-PCR assay. ( B ) Protein-protein interaction (PPI) network of E2f8, Ccna2, Rrm2, Cdk1, Cdc45, Ccne1, and E2f7. ( C ) E2f8 protein levels. β-actin as an internal reference. ( D ) Statistical analysis of the E2F8 protein levels (mean ± SD, n = 3). ( E ) The mRNA expression of Ccnd1, Cdk2, Cdk4, p21, and p27 were validated by qRT-PCR assay. ( F ) Cyclin D1, Cyclin E1, Cdk2, Cdk4, P21 and P27 protein levels. GAPDH as internal reference. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control group.

Article Snippet: Antibodies against Cleaved caspase-3, β-actin, GAPDH, and anti-mouse HRP, anti-rabbit HRP were purchased from Zen Bioscience were purchased from Zen-bio (Chengdu, China).

Techniques: Expressing, Quantitative RT-PCR, Control