Journal: Frontiers in Immunology

Article Title: HTNV Sensitizes Host Toward TRAIL-Mediated Apoptosis—A Pivotal Anti-hantaviral Role of TRAIL

doi: 10.3389/fimmu.2020.01072

Figure Lengend Snippet: HTNV induces TRAIL-dependent apoptosis in primary HUVECs. HUVECs were infected with/without HTNV 76-118 (MOI = 1) for 2 h and followed by qRT-PCR and Western blot at different days to measure TRAIL-related apoptosis factors. (A) HTNV S, TRAIL, DcR1, DcR2, caspase-8, and caspase-3 mRNA in HTNV-infected HUVECs. (B) HTNV NP and TRAIL-related apoptosis proteins expression in HTNV-infected HUVECs. (C) TRAIL-DR4/DR5 and (D) caspase-9 mRNA and protein expression during HTNV infection. The mRNA results shown are the average of three replicates; values represent the mean ± SD ( * p < 0.05; ** p < 0.01; *** p < 0.001). Proteins were quantified using carestream software and compared with the normal control group of 2 dpi. Data were from one of three experiments with similar results. The numbers represented the relative density of the bands relative to the corresponding control. (E) Histogram showing DR4, DR5, DcR1, and DcR2 surface expression on HTNV-infected and uninfected HUVECs at 2 dpi. White histograms indicate fluorescent labeling with receptor-specific monoclonal antibodies, and gray histograms show background labeling with isotype-matched control antibody in the same group. The experiment was repeated three times and data represent one of three separate experiments. (F) Data showing the MFI of DR4, DR5, DcR1, and DcR2 surface expression on HTNV-infected relative to the uninfected HUVECs at 2 dpi. The MFI of TRAIL receptors was assessed by FlowJo software. The experiment was repeated three times. The dots represented the average of each experiment and data were analyzed by a two-tailed, two-sample student t -test ( p -values were indicated).

Article Snippet: Each membrane was, respectively, probed with a polyclonal antibody against TRAIL protein (Abcam ab2435, 1:1,000), human DR4 (Flarebio CSB-PA002191, 1:1,000), human DR5 (Flarebio CSB-PA08165A0Rb, 1:1,000), human DcR1 (Abcam ab133658, 1:2,000), human DcR2 (Cusabio E0425R1, 1:1,000), human caspase-3 (Beyotime AC033, 1:1,000), human caspase-8 (Beyotime AC056, 1:1,000), human caspase-9 (Beyotime AC062, 1:1,000), human cleaved-PARP (Beyotime AP102, 1:1,000), a mouse monoclonal antibody against GAPDH (Tianjin Sungene Biotech DKM9002T, 1:5,000), and IRF3 (ABclonal Q14653, 1:1,000), IRF7 (ABclonal Q92985, 1:1,000).

Techniques: Infection, Quantitative RT-PCR, Western Blot, Expressing, Software, Control, Labeling, Bioprocessing, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: HTNV Sensitizes Host Toward TRAIL-Mediated Apoptosis—A Pivotal Anti-hantaviral Role of TRAIL

doi: 10.3389/fimmu.2020.01072

Figure Lengend Snippet: The hantaviral structural proteins affect the transcriptional activity of the promoters of TRAIL/TRAIL-Rs. Firefly luciferase reporter plasmids (400 ng) containing TRAIL, DR4, DR5, DcR1, or DcR2 promoter and hantaviral structural plasmids (400 ng) including pSicoR (vector), pSicoR-S, or pSicoR-M were co-transfected into HEK293T cells to detect the promoter activity. Luciferase activity represented the effect of hantaviral structural proteins on the promoters of TRAIL/TRAIL-Rs. The data were presented with the mean ± SD from three independent experiments of relative luciferase activity ( * p < 0.05; ** p < 0.01).

Article Snippet: Each membrane was, respectively, probed with a polyclonal antibody against TRAIL protein (Abcam ab2435, 1:1,000), human DR4 (Flarebio CSB-PA002191, 1:1,000), human DR5 (Flarebio CSB-PA08165A0Rb, 1:1,000), human DcR1 (Abcam ab133658, 1:2,000), human DcR2 (Cusabio E0425R1, 1:1,000), human caspase-3 (Beyotime AC033, 1:1,000), human caspase-8 (Beyotime AC056, 1:1,000), human caspase-9 (Beyotime AC062, 1:1,000), human cleaved-PARP (Beyotime AP102, 1:1,000), a mouse monoclonal antibody against GAPDH (Tianjin Sungene Biotech DKM9002T, 1:5,000), and IRF3 (ABclonal Q14653, 1:1,000), IRF7 (ABclonal Q92985, 1:1,000).

Techniques: Activity Assay, Luciferase, Plasmid Preparation, Transfection

Journal: Frontiers in Immunology

Article Title: HTNV Sensitizes Host Toward TRAIL-Mediated Apoptosis—A Pivotal Anti-hantaviral Role of TRAIL

doi: 10.3389/fimmu.2020.01072

Figure Lengend Snippet: Exogenous rTRAIL inhibits HTNV infection by enhancing caspase-8-dependent apoptosis. HUVECs were infected with HTNV76-118 (MOI = 1) for 2 h, treated with/without rTRAIL (40 ng/mL), and then followed by qRT-PCR and Western blot at different days to measure TRAIL-related apoptosis factors and HTNV. (A) HTNV S, TRAIL DR4, DR5, caspase-8, and caspase-3 mRNA expression. (B) HTNV NP and TRAIL-related apoptosis proteins. (C) DcR1/DcR2 mRNA and protein. The mRNA results shown are the average of three replicates and values represented the mean ± SD ( * p < 0.05; ** p < 0.01; *** p < 0.001). The protein result represents one of three similar experiments. Numbers represent the relative density of the bands relative to the internal control. (D) Immunofluorescence images and (E) statistical analysis of TUNEL assay on HTNV-infected HUVECs with rTRAIL treatment. Cells were stained for virus infection [NP (sred)], for apoptosis [TUNEL (green)], and for nucleus [Hoechst (blue)]. Images data showed one of three independent experiments with similar results.

Article Snippet: Each membrane was, respectively, probed with a polyclonal antibody against TRAIL protein (Abcam ab2435, 1:1,000), human DR4 (Flarebio CSB-PA002191, 1:1,000), human DR5 (Flarebio CSB-PA08165A0Rb, 1:1,000), human DcR1 (Abcam ab133658, 1:2,000), human DcR2 (Cusabio E0425R1, 1:1,000), human caspase-3 (Beyotime AC033, 1:1,000), human caspase-8 (Beyotime AC056, 1:1,000), human caspase-9 (Beyotime AC062, 1:1,000), human cleaved-PARP (Beyotime AP102, 1:1,000), a mouse monoclonal antibody against GAPDH (Tianjin Sungene Biotech DKM9002T, 1:5,000), and IRF3 (ABclonal Q14653, 1:1,000), IRF7 (ABclonal Q92985, 1:1,000).

Techniques: Infection, Quantitative RT-PCR, Western Blot, Expressing, Control, Immunofluorescence, TUNEL Assay, Staining, Virus

Journal: Frontiers in Immunology

Article Title: HTNV Sensitizes Host Toward TRAIL-Mediated Apoptosis—A Pivotal Anti-hantaviral Role of TRAIL

doi: 10.3389/fimmu.2020.01072

Figure Lengend Snippet: shRNA-mediated knockdown and rescue of TRAIL influence HTNV replication and apoptosis in HUVECs. HUVEC TRAIL−KD cells were infected with HTNV76-118 (MOI = 1) for 2 h, treated with/without rTRAIL (40 ng/mL), and then followed by qRT-PCR and Western blot at different days to measure TRAIL-related apoptosis factors and HTNV. The mRNA results shown were the average of three replicates. Values represent the mean ± SD ( * p < 0.05; ** p < 0.01; *** , p 0.001). (A) HTNV S mRNA, (B) TRAIL mRNA, (C) DR4 mRNA, (D) DR5 mRNA, (E) DcR1 mRNA, (F) DcR2 mRNA, (G) caspase-8 mRNA, and (H) caspase-3 mRNA. (I) HTNV NP and TRAIL-related apoptosis proteins in HTNV-infected HUVEC TRAIL−KD cells at 2, 3, and 4 dpi. (J) HTNV NP and TRAIL-related apoptosis proteins of HUVEC TRAIL−KD cells with rTRAIL rescue. Proteins were quantified using carestream software and compared with the mock group of 2 dpi; the data shown represents one of three similar independent experiments. The numbers represent the relative density of the bands relative to the corresponding control.

Article Snippet: Each membrane was, respectively, probed with a polyclonal antibody against TRAIL protein (Abcam ab2435, 1:1,000), human DR4 (Flarebio CSB-PA002191, 1:1,000), human DR5 (Flarebio CSB-PA08165A0Rb, 1:1,000), human DcR1 (Abcam ab133658, 1:2,000), human DcR2 (Cusabio E0425R1, 1:1,000), human caspase-3 (Beyotime AC033, 1:1,000), human caspase-8 (Beyotime AC056, 1:1,000), human caspase-9 (Beyotime AC062, 1:1,000), human cleaved-PARP (Beyotime AP102, 1:1,000), a mouse monoclonal antibody against GAPDH (Tianjin Sungene Biotech DKM9002T, 1:5,000), and IRF3 (ABclonal Q14653, 1:1,000), IRF7 (ABclonal Q92985, 1:1,000).

Techniques: shRNA, Knockdown, Infection, Quantitative RT-PCR, Western Blot, Software, Control

Journal: Cell Death & Disease

Article Title: Ischemic tolerance modulates TRAIL expression and its receptors and generates a neuroprotected phenotype

doi: 10.1038/cddis.2014.286

Figure Lengend Snippet: Effect of tMCAO/PC followed by 24 h of reperfusion on either TRAIL and its receptor's protein expression, as well as on TNF- α , FasL, and their respective receptors. ( a ) Representative blots of DR4, DR5, TRAIL, DcR1, and DcR2 protein expression in sham-operated animals (SHAM) or after 100 min tMCAO (tMCAO), 30 min tMCAO (PC), and 30 min PC, followed, 72 h later, by 100 min tMCAO (PC+tMCAO) in the ipsilateral temporoparietal cortex after 24 h of reperfusion. Tubulin blots: β -tubulin (respective controls). ( b ) Representative blots of TNFR1, TNF- α , Fas, and FasL protein expression in sham-operated animals (SHAM) and after 100 min tMCAO (tMCAO), 30 min tMCAO (PC), and 30 min PC, followed, 72 h later, by 100 min tMCAO (PC+tMCAO) in the ipsilateral temporoparietal cortex after 24 h of reperfusion. Tubulin blots: β -tubulin (respective controls)

Article Snippet: The membranes were blocked with 5% milk in PBST and then incubated overnight at 4 °C with a rabbit anti-DR4 polyclonal antibody (ProSci Inc., Poway, CA, USA) or a rabbit anti-DR5 polyclonal antibody (Abcam, Cambridge, UK) or a rabbit anti-TRAIL polyclonal antibody (Abcam) or a rabbit anti-DcR1 (ProSci) polyclonal antibody or a rabbit anti-DcR2 polyclonal antibody (Abcam) or a rabbit anti-TNFRI polyclonal antibody (Abcam) or a rabbit anti-TNF alpha polyclonal antibody (Novus Biologicals, Littleton, CO, USA) or a mouse anti-Fas monoclonal antibody (BD Biosciences, San Jose, CA, USA) or a rabbit anti-FasL polyclonal antibody (Abcam) or a mouse anti-Caspase-8 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) or a rabbit anti-Caspase-3 monoclonal antibody (Cell Signaling Technology) or a mouse anti-Phospho-JNK monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a rabbit anti-JNK polyclonal antibody (Santa Cruz Biotechnology) or a rabbit anti-Phospho-Akt monoclonal antibody (Cell Signaling Technology) or a rabbit anti-Akt monoclonal antibody (Cell Signaling Technology) and horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibody (Amersham Life Science). β -Tubulin (Santa Cruz Biotechnology) was used as an internal control to validate the right amount of protein loaded in the gels.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Ischemic tolerance modulates TRAIL expression and its receptors and generates a neuroprotected phenotype

doi: 10.1038/cddis.2014.286

Figure Lengend Snippet: Effect of 100 min of transient brain ischemia (tMCAO), ischemic PC, and PC followed 72 h later by tMCAO (PC+tMCAO) on DR4 expression. Confocal microscopic images displaying NeuN (a–l) (green), DR4 (b–m) (red), and Merge (c–n) (yellow) in the brain peri-ischemic region of rats after 5 h ( A ), 24 h ( B ), and 72 h ( C ) of reperfusion. A representative brain slice cartoon indicating the area of interest is on the left top of the figure. Scale bars in a–i: 50 μ m

Article Snippet: The membranes were blocked with 5% milk in PBST and then incubated overnight at 4 °C with a rabbit anti-DR4 polyclonal antibody (ProSci Inc., Poway, CA, USA) or a rabbit anti-DR5 polyclonal antibody (Abcam, Cambridge, UK) or a rabbit anti-TRAIL polyclonal antibody (Abcam) or a rabbit anti-DcR1 (ProSci) polyclonal antibody or a rabbit anti-DcR2 polyclonal antibody (Abcam) or a rabbit anti-TNFRI polyclonal antibody (Abcam) or a rabbit anti-TNF alpha polyclonal antibody (Novus Biologicals, Littleton, CO, USA) or a mouse anti-Fas monoclonal antibody (BD Biosciences, San Jose, CA, USA) or a rabbit anti-FasL polyclonal antibody (Abcam) or a mouse anti-Caspase-8 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) or a rabbit anti-Caspase-3 monoclonal antibody (Cell Signaling Technology) or a mouse anti-Phospho-JNK monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a rabbit anti-JNK polyclonal antibody (Santa Cruz Biotechnology) or a rabbit anti-Phospho-Akt monoclonal antibody (Cell Signaling Technology) or a rabbit anti-Akt monoclonal antibody (Cell Signaling Technology) and horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibody (Amersham Life Science). β -Tubulin (Santa Cruz Biotechnology) was used as an internal control to validate the right amount of protein loaded in the gels.

Techniques: Expressing, Slice Preparation