Journal: Arthritis and rheumatism

Article Title: Mechanisms and clinical relevance of TRAIL-triggered responses in the synovial fibroblasts of patients with rheumatoid arthritis.

doi: 10.1002/art.30181

Figure Lengend Snippet: Figure 4. Rheumatoid arthritis fibroblast-like synoviocytes (RA FLS) produce low levels of the inflammatory cytokines interleukin-6 (IL-6) and IL-8 when stimulated with TRAIL. RA FLS (n 5) were treated with TRAIL (1 nM) or tumor necrosis factor (TNF; 0.5 nM) for 72 hours, and supernatants were analyzed for IL-6 (A), IL-8 (B), and RANTES (C) secretion by enzyme-linked immunosorbent assay. Cy- tokine levels were analyzed using Wilcoxon’s test. Values are the mean SEM. NS not stimulated.

Article Snippet: Serum levels of TRAIL and OPG were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) (Quantikine human OPG ELISA Kit [R&D Systems] and a human TRAIL ELISA [Diaclone]).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Acta Neuropathologica Communications

Article Title: TSPO acts as an immune resistance gene involved in the T cell mediated immune control of glioblastoma

doi: 10.1186/s40478-023-01550-9

Figure Lengend Snippet: TSPO protects GB cells from TRAIL-induced apoptosis. a Resazurin assay to analyze the impact of TNFα, IFNγ, and FasL treatment on the viability of TSPO ± BTIC13 cells. Cells were treated for 48 h and fluorescence was normalized to untreated condition for control or TSPO shRNA-transduced cells. b Western blot analysis of total/cleaved caspase-9, total caspase-3, and total/cleaved PARP1 in TSPO ± BTIC13 cells upon 1, 3, and 6 h treatment with 50 ng/ml TNFα, IFNγ, and FasL (cl = cleaved; n.d. = not detected). c Resazurin assay to analyze the impact of TRAIL treatment on the viability of TSPO ± BTIC13 cells. Dose–response curves indicate normalized %-cell viability versus log-transformed 50 ng/ml TRAIL concentration. EC 50 values were determined by nonlinear curve fitting. d Western blot analysis of total/cleaved caspase-9/-3 and PARP1 in TSPO ± BTIC13 cells upon 1 and 3 h treatment with TRAIL. e, f Resazurin assay to analyze the impact of ( e ) 50 ng/ml TNFα, IFNγ, and FasL and ( f ) TRAIL treatment on the viability of TSPO ± BTIC13 clones. g Luciferase-based caspase-3/-7 assay to measure caspase-3/-7 activation in TSPO ± BTIC13 clones after 4 h treatment with 50 ng/ml TRAIL. h Real-time cytotoxicity assay to analyze TRAIL-induced caspase-3/-7 activation over 24 h in BTIC13 clones. The graphs show the total apoptotic tumor cell area (green object area) per well. i, j Western blot analysis of total/cleaved caspase-9/-3 and PARP1 in TSPO ± ( i ) BTIC13 clones upon 1 and 3 h treatment with 50 ng/ml TRAIL and ( j ) BTIC129 cells upon 6 h treatment with 50 ng/ml TNFα, FasL and TRAIL. k Luciferase-based caspase-3/-7 assay to measure caspase-3/-7 activation in TSPO ± BTIC129 cells after 4 h treatment with TRAIL. l Real-time cytotoxicity assay to analyze TRAIL-induced caspase-3/-7 activation over 24 h in BTIC129 cells. The graphs show the total apoptotic tumor cell area (green object area) per well. Representative data of at least two independent experiments. e, f Cumulative data of three independent experiments. Values represent the mean of triplicates ± SD. P-value was calculated using two-tailed Student`s t-test (* = P < 0.05, ** = P < 0.01, *** = P < 0.005, **** = P < 0.001)

Article Snippet: Supernatants of T cell-tumor cell co-cultures and anti-CD3/CD28 activated T cells were analyzed for the detection of IFNγ (Human IFN-γ ELISA Set, BD OptEIA #555142), Tumor necrosis factor alpha (TNFα) (Human TNF ELISA Set, BD OptEIA #555212), Granzyme B (Human Granzyme B ELISA development kit, Mabtech #3485-1H-20) and TRAIL (human TRAIL/TNFSF10 DuoSet ELISA, R&D Systems, #DY375-05).

Techniques: Resazurin Assay, Fluorescence, Control, shRNA, Western Blot, Transformation Assay, Concentration Assay, Clone Assay, Luciferase, Activation Assay, Cytotoxicity Assay, Two Tailed Test

Journal: Acta Neuropathologica Communications

Article Title: TSPO acts as an immune resistance gene involved in the T cell mediated immune control of glioblastoma

doi: 10.1186/s40478-023-01550-9

Figure Lengend Snippet: TSPO regulates the expression of genes associated with apoptosis-resistance. a Principal component analysis (PCA) of RNA-Seq gene expression profiles from TSPO ± BTIC13. b Volcano Plot highlighting differentially expressed genes in TSPO-deficient BTIC13 (fold change ≥ 2, normalized counts per million > 2, false discovery rate (FDR) ≤ 0.05, labelled blue (downregulated) and red (upregulated)). c Heatmap of expression changes of DEGs associated with GO term: GOBP_REGULATION_OF_CELL_DEATH (GO:0010941) in TSPO ± BTIC13. Anti-apoptotic genes are indicated with a blue arrow. d Correlation between the expression of TSPO and CCDN2 , B3GALT2 , CEMIP , APOBEC3G , PI3 , and SLPI at single-cell level . R indicates Pearson`s correlation coefficient. e RT-qPCR analysis of PI3 and SLPI mRNA expression in control medium (CM) or TRAIL treated TSPO ± BTIC13. Results are presented as fold change compared to the CM condition of TSPO-proficient cells after β-actin mRNA normalization. f Luciferase-based caspase-3/-7 assay to measure caspase-3/-7 activation in PI3/SLPI downregulated BTIC13 upon 4 h co-culture with FluTC or treatment with 50 ng/ml TRAIL. g Real-time cytotoxicity assay to analyze TRAIL-induced caspase-3/-7 activation over 24 h in BTIC13 cells upon PI3 and SLPI double knockdown. The graphs show the total apoptotic tumor cell area (green object area) per well. h Western blot analysis of total/cleaved caspase-9/-3 and PARP1 in BTIC13 transfected with control or PI3-SLPI-specific siRNAs upon treatment with 50 ng/ml TRAIL. g, h Values represent the mean of triplicates ± SD. P-value was calculated using two-tailed Student`s t-test (* = P < 0.05, ** = P < 0.01, *** = P < 0.005, **** = P < 0.001)

Article Snippet: Supernatants of T cell-tumor cell co-cultures and anti-CD3/CD28 activated T cells were analyzed for the detection of IFNγ (Human IFN-γ ELISA Set, BD OptEIA #555142), Tumor necrosis factor alpha (TNFα) (Human TNF ELISA Set, BD OptEIA #555212), Granzyme B (Human Granzyme B ELISA development kit, Mabtech #3485-1H-20) and TRAIL (human TRAIL/TNFSF10 DuoSet ELISA, R&D Systems, #DY375-05).

Techniques: Expressing, RNA Sequencing, Gene Expression, Quantitative RT-PCR, Control, Luciferase, Activation Assay, Co-Culture Assay, Cytotoxicity Assay, Knockdown, Western Blot, Transfection, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: TRAIL Mediates Neuronal Death in AUD: A Link between Neuroinflammation and Neurodegeneration

doi: 10.3390/ijms22052547

Figure Lengend Snippet: Ethanol-induced neurodegeneration in primary organotypic brain slice culture involves TRAIL. ( A ) Organotypic brain slice cultures (OBSCs) were treated with ethanol (100 mM) for 4 days. TRAIL was measured by ELISA 16 h into withdrawal prior to onset of neuronal cell death. Ethanol caused a 36% increase in TRAIL in slice tissue. n = 9 control, 3 ethanol slices. ( B ) OBSCs were treated with ethanol (100 mM) for 24–72 h. TRAIL-R1/DR4 and TRAIL R2/DR5 were measured by Western blot. Ethanol caused a 1.55-fold increase in TRAIL-R1 protein levels at 24 h and a 1.66-fold increase at 72 h (* p < 0.05). Ethanol caused a trend toward a 1.2-fold increase in TRAIL-R2 protein levels at 72 h ( p = 0.09, n = 9 control and 3 ethanol). Images of blots at 72 h of EtOH treatment are shown. ( C , D ) The ability of ethanol pretreatment to enhance TRAIL neurotoxicity was assessed. OBSCs were treated with ethanol for 48 h, which was followed by addition of recombinant TRAIL (0.75 µg/mL) for 24 h. Neurodegeneration was assessed by propidium iodide (PI) uptake. ( C ) Ethanol and TRAIL alone caused a moderate amount of neurodegeneration. However, ethanol pretreatment caused a robust, synergistic enhancement in neurodegeneration due to TRAIL. ANOVA: F3.34 = 7.2, p = 0.0007. *** p < 0.001 vs. control, # p < 0.05 vs. TRAIL alone, Sidak’s multiple comparisons test. n = 9–10 slices/group ( D ) Representative image showing enhancement of TRAIL neurodegeneration by ethanol pretreatment. ( E ) The role of TRAIL in ethanol-induced neurodegeneration was assessed using the TRAIL monoclonal antibody (αTRAIL mAb). OBSC was treated with ethanol (100 mM) for 72 h followed by ethanol withdrawal (EW) for 24 h ± rat αTRAIL mAb (0.5 µg/mL) or rat immunoglobulin G (IgG) isotype control. EW caused significant neurodegeneration, which was completely abolished by αTRAIL mAb. ANOVA: F4.42 = 9.3, p < 0.0001. **** p < 0.0001 vs. IgG alone control, ††† p < 0.001 vs. EW + IgG, Sidak’s multiple comparisons test. n = 7–16 slices per group. ( F ) Representative images showing inhibition of EW-induced neurodegeneration by αTRAIL mAb. Fluorescent PI images were converted to black and white images for ease of visualization.

Article Snippet: Anti-TLR7 (NBP2-24906)—Novus Biologicals, Littleton, CO; anti-NeuN (MAB377), anti-GAPDH (ab2302), and Fluoro-Jade B—EMD Millipore (Temecula, CA, USA); anti-p-NFκB p65 (sc-101749)—Santa Cruz Biotechnology (Dallas, TX, USA); anti-HMGB1 (ab18256), anti-glial fibrillary acidic protein (GFAP) (ab4648), anti-DR4 (ab8414), anti-DR5 (ab8416), and anti-Iba1 (ab5076)—Abcam (Cambridge, MA, USA); cleaved caspase-3 (9661L)—Cell Signaling Technology (Danvers, MA, USA); rat anti-TRAIL antibody (550320) and rat IgG2a κ isotype control (554687)—BD Biosciences, (San Jose, CA, USA); anti-human TLR7 ELISA (MBS263437)—MyBioSource (San Diego, CA, USA); anti-rat TRAIL ELISA (LS-F23243)—LifeSpan BioSciences Inc. (Seattle, WA, USA); anti-human TRAIL ELISA (DY375)—R&D Systems (Minneapolis, MN, USA).

Techniques: Slice Preparation, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Recombinant, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: TNFα and IFNβ do not trigger cell apoptosis. A, poly(I-C) up-regulated the expression of TNFα in immortalized HUVECs. Cells were treated with the indicated concentrations of poly(I-C) for 24 h. The supernatant was harvested to detect the TNFα secretion by ELISA. Data are represented as mean ± S.D. of triplicates. *, p < 0.05 compared with the medium control. B, TNFα (37 °C for 24 h) did not induce cell apoptosis in immortalized HUVECs. C, TNFα (50 ng/ml) induced NF-κB signaling in immortalized HUVECs. D, TNFα neutralization did not inhibit poly(I-C)-induced cell apoptosis in immortalized HUVECs. E, IFNβ (37 °C for 24 h) did not induce cell apoptosis in immortalized HUVECs. F, IFNβ neutralization did not inhibit poly(I-C)-induced cell apoptosis in immortalized HUVECs.

Article Snippet: Human TRAIL ELISA kit was purchased from Boster Biochemicals (Wuhan, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control, Neutralization

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: TRAIL-DR4/5 and Noxa trigger the extrinsic and intrinsic pathways, respectively. A and B, RT-PCR results show that poly(I-C) (37 °C for 24 h) up-regulated the gene expression of TRAIL, DR4, and DR5 in immortalized (A) and 1 μg/ml poly(I-C) pretreated primary (B) HUVECs. C, poly(I-C) up-regulated the protein expression of TRAIL in primary HUVECs. Cells, pretreated with 1 μg/ml poly(I-C), were re-treated with the indicated concentrations of poly(I-C) for 24 h. TRAIL in the cell lysates was assayed by ELISA. *, p < 0.05 compared with the control. D, TRAIL neutralization repressed the cell apoptosis induced by poly(I-C) in immortalized HUVECs. *, p < 0.05 compared with the poly(I-C) treatment group. E and F, RT-PCR results show the effect of poly(I-C) (37 °C for 24 h) on the gene expression of Bcl-2 and Noxa in immortalized (E) and primary (F) HUVECs. G and H, Western blot results show the effect of poly(I-C) on the protein expression of Bcl-2 and Noxa in immortalized (G) and primary (H) HUVECs. I, inhibition of TLR3 repressed the poly(I-C)-induced down-regulation of Bcl-2 and up-regulation of Noxa in immortalized HUVECs. Cells were transiently transfected with human TLR3 shRNA plasmid and then treated with 2 μg/ml poly(I-C) for 24 h. The protein expression of TLR3, Bcl-2 and Noxa was detected by Western blot.

Article Snippet: Human TRAIL ELISA kit was purchased from Boster Biochemicals (Wuhan, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Control, Neutralization, Western Blot, Inhibition, Transfection, shRNA, Plasmid Preparation

Journal: Internal and Emergency Medicine

Article Title: Serum TRAIL predicts severity and prognosis in patients with community-acquired pneumonia: a prospective cohort study

doi: 10.1007/s11739-022-03086-7

Figure Lengend Snippet: The levels of circulatory TRAIL in CAP patients with different severity. A-F The levels of circulatory TRAIL were detected through ELISA in CAP patients with different severity scores. A The levels of circulatory TRAIL in CAP patients with different CRB-65 scores. B The levels of circulatory TRAIL in CAP patients with different CURB-65 scores. C The levels of circulatory TRAIL in CAP patients with different SMART-COP scores. D The levels of circulatory TRAIL in CAP patients with different CURXO scores. E The levels of circulatory TRAIL in CAP patients with different PSI scores. F The levels of circulatory TRAIL in CAP patients with different APACHE II scores. * P < 0.5, ** P < 0.01

Article Snippet: TRAIL (CSB-E13164h) and interleukin-1β (IL-1β) (CSB-E08053h) ELISA kits were purchased from Cusabio, Wuhan, China ( https://www.cusabio.com/ ).

Techniques: Enzyme-linked Immunosorbent Assay