Journal: Cancer science

Article Title: Aclarubicin enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis through death receptor 5 upregulation.

doi: 10.1111/j.1349-7006.2011.02150.x

Figure Lengend Snippet: Fig. 1. Aclarubicin (ACR) more effectively induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis than doxorubicin (DOX). (a) Human acute lymphoblastic leukemia Jurkat cells were treated with DMSO alone (control) or the indicated concentrations of ACR (r) or DOX ( ). After incubation for 72 h, viable cells were counted using Cell Counting Kit-8 (Dojindo). Data represent means ± SD of three determinations. *P < 0.05 (vs control). (b) Jurkat cells were treated with 120 nM ACR or 120 nM DOX for 24 h, and 50 ng ⁄ mL of TRAIL for an additional 12 h. Cell viability was measured using the ViaCount Assay (Guava Technologies). Data represent means ± SD of three determinations. *P < 0.05 (c) Jurkat cells were treated with 120 nM ACR or 120 nM DOX for 24 h, then 50 ng ⁄ mL of TRAIL for an additional 12 h. Hypodiploid cells (Sub-G1) were measured by flow cytometry. Data represent means ± SD of three determinations. *P < 0.05 (vs control or TRAIL or ACR or DOX). (d) Western blotting for death receptor 5 (DR5) was performed with lysate of cells treated for 24 h with ACR or DOX at indicated concentrations. These two band patterns of DR5 protein are consistent with the results of a previous report.(31) b-actin is shown as a loading control. (e) The cells were treated with 120 nM of ACR or DOX for 24 h, and luciferase activity from pDR5PF with a 2.5-kbp DR5 promoter and the luciferase gene was measured. Relative luciferase activity was standardized by the amount of protein. pGVB2 is a control vacant plasmid. Data represent means ± SD of three determinations.

Article Snippet: A rabbit polyclonal anti-DR5 antibody (Prosci, Poway, CA, USA), mouse monoclonal anti-Bid, and anti-caspase-8, anti-caspase-9 and anti-caspase-10 antibodies (MBL, Nagoya, Japan), a mouse monoclonal anti-pro-caspase-3 antibody (Immunotech, Marseile, France), a Horinaka et al. rabbit monoclonal cleaved caspase-3 antibody (Cell Signaling, Beverly, MA, USA) and a mouse monoclonal anti-b-actin antibody (Sigma) were used as the primary antibodies.

Techniques: Control, Incubation, Cell Counting, Cytometry, Western Blot, Luciferase, Activity Assay, Plasmid Preparation

Journal: Cancer science

Article Title: Aclarubicin enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis through death receptor 5 upregulation.

doi: 10.1111/j.1349-7006.2011.02150.x

Figure Lengend Snippet: Fig. 2. Aclarubicin (ACR) upregulates mRNA and protein expression of death receptor (DR) 5. (a) Jurkat cells were treated with 120 nM ACR for 24 h and total RNA was extracted. The RNase protection assay was carried out as described in the Materials and Methods. Probes not digested by RNase are also shown. As a control, yeast tRNA was used instead of total RNA. L32 and GAPDH are control genes to show that equal amounts of total RNA were used. (b) Quantitative real-time RT-PCR of DR5 mRNA in Jurkat cells treated with 120 nM ACR or 120 nM doxorubicin (DOX) for 24 h. The internal control was 18S rRNA. Data represent means ± SD of three determinations. *P < 0.05 (vs DMSO) (c) Jurkat cells were treated with 120 nM ACR for the periods indicated. Total cell lysate was used for western blotting of DR5. b-actin is shown as a loading control. (d) Jurkat cells were treated with 120 nM ACR for 24 h. Cell lysate was separated into subcellular fractions. Western blotting for DR5 was performed with cytosolic and membrane fractions. The gel was stained with coomassie-brilliant blue (CBB) and a loading control is shown below the western blot. DcR, decoy receptor; RIP, receptor-interacting protein; TNFR, tumor necrosis factor receptor; TRADD, TNFR1- associated death domain; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

Article Snippet: A rabbit polyclonal anti-DR5 antibody (Prosci, Poway, CA, USA), mouse monoclonal anti-Bid, and anti-caspase-8, anti-caspase-9 and anti-caspase-10 antibodies (MBL, Nagoya, Japan), a mouse monoclonal anti-pro-caspase-3 antibody (Immunotech, Marseile, France), a Horinaka et al. rabbit monoclonal cleaved caspase-3 antibody (Cell Signaling, Beverly, MA, USA) and a mouse monoclonal anti-b-actin antibody (Sigma) were used as the primary antibodies.

Techniques: Expressing, Rnase Protection Assay, Control, Quantitative RT-PCR, Western Blot, Membrane, Staining

Journal: Cancer science

Article Title: Aclarubicin enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis through death receptor 5 upregulation.

doi: 10.1111/j.1349-7006.2011.02150.x

Figure Lengend Snippet: Fig. 3. Combination of aclarubicin (ACR) and tumor necrosis factor- related apoptosis-inducing ligand (TRAIL) induces caspase-dependent apoptosis through the TRAIL receptors. (a) Jurkat cells were treated with ACR and ⁄ or TRAIL with or without caspase inhibitors. The population of sub-G1 cells was analyzed by flow cytometry. Data represent means ± SD of three determinations. *P < 0.05 (vs the combined treatment with ACR and TRAIL). (b) Jurkat cells were treated with ACR at the indicated concentrations for 24 h, followed by 50 ng ⁄ mL TRAIL for an additional 12 h with or without the death receptor 5 (DR5) ⁄ Fc chimera. Western blotting for the proteins indicated was carried out. b-actin is shown as a loading control.

Article Snippet: A rabbit polyclonal anti-DR5 antibody (Prosci, Poway, CA, USA), mouse monoclonal anti-Bid, and anti-caspase-8, anti-caspase-9 and anti-caspase-10 antibodies (MBL, Nagoya, Japan), a mouse monoclonal anti-pro-caspase-3 antibody (Immunotech, Marseile, France), a Horinaka et al. rabbit monoclonal cleaved caspase-3 antibody (Cell Signaling, Beverly, MA, USA) and a mouse monoclonal anti-b-actin antibody (Sigma) were used as the primary antibodies.

Techniques: Cytometry, Western Blot, Control

Journal: Cancer science

Article Title: Aclarubicin enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis through death receptor 5 upregulation.

doi: 10.1111/j.1349-7006.2011.02150.x

Figure Lengend Snippet: Fig. 4. Aclarubicin (ACR) induces death receptor 5 (DR5) expression and enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in the lung cancer cell line A549. (a) Western blotting for DR5. Human lung cancer A549 cells were treated with ACR at the concentrations indicated for 24 h. b-actin is shown as a loading control. (b) A549 cells were treated with ACR or doxorubicin (DOX) at the concentrations indicated for 24 h, followed by 50 ng ⁄ mL TRAIL for an additional 12 h. The sub-G1 population was analyzed by flow cytometry. Data represent means ± SD of three determinations. *P < 0.05 (vs control or TRAIL or ACR or DOX).

Article Snippet: A rabbit polyclonal anti-DR5 antibody (Prosci, Poway, CA, USA), mouse monoclonal anti-Bid, and anti-caspase-8, anti-caspase-9 and anti-caspase-10 antibodies (MBL, Nagoya, Japan), a mouse monoclonal anti-pro-caspase-3 antibody (Immunotech, Marseile, France), a Horinaka et al. rabbit monoclonal cleaved caspase-3 antibody (Cell Signaling, Beverly, MA, USA) and a mouse monoclonal anti-b-actin antibody (Sigma) were used as the primary antibodies.

Techniques: Expressing, Western Blot, Control, Cytometry

Journal: Journal of Neuroinflammation

Article Title: Transcriptomics and translatomics identify a robust inflammatory gene signature in brain endothelial cells after ischemic stroke

doi: 10.1186/s12974-023-02888-6

Figure Lengend Snippet: Ischemia-induced upregulation of the expression of certain immunomodulatory membrane receptors as assessed by flow cytometry. A Gating strategy for flow cytometry analysis. B Quantification of the % of CD31 + endothelial cells expressing the receptors in the contralateral (contra) and ipsilateral (ipsi, ischemic) hemispheres 24h post-ischemia ( n = 6 per group) by flow cytometry. Ischemia increased the % of CD31 + cells expressing CD262 ( Tnfrsf10b) (* p = 0.0156), CD155 ( Pvr ) (* p = 0.0156), and CD146 ( Mcam ) (* p = 0.0156). Changes in the % of CD31 + cells expressing CD354 ( Trem1 ) ( p = 0.1562) were not significant. Analysis was performed with the Wilcoxon matched-pairs signed rank test. C , D Immunofluorescence for CD146 (red in C ) and CD155 (green in D ) in the brain vessels (Glut1 + ) of sham-operated and ischemic mice at 1- and 4-day post-ischemia. Nuclei are stained with DAPI (blue). Scale bar: 20 μm. Images in the bottom row are magnifications of the squares shown in the merged images at day 4 post-ischemia. Quantification of mean fluorescence intensity (MFI) of CD146 and CD155 in brain vessels (number of vessels ranging from 145 to 250 obtained from 3 to 5 mice per group) **** p < 0.0001 (Kruskal–Wallis and Dunn’s tests)

Article Snippet: Cells were incubated during 30 min at 4 °C with the following primary antibodies: CD31 (clone 390, PE-Cyanine7, #25-0311-82, eBioscience, RRID:AB_2716949; or clone 390, BV421, #102423, Biolegend, RRID:AB_2562186), CD11b (clone M1/70, PE, #553311, BD Pharmingen, RRID:AB_394775), CD45 (clone 30-F11, FITC, #553080, BD Horizon, RRID:AB_394610), CD155 (clone TX56, BV421, #131517, Biolegend, RRID:AB_2716159), CD354 (clone 174031, BV711, #747902, BD Pharmingen, RRID:AB_2872364), CD146 (clone ME-9F1, PE-Cyanine7, #134713, Biolegend, RRID:AB_2563108), CD262 (clone MD5-1-3C2, PerCP-Vio700, #130-105-697, Miltenyi Biotec, RRID:AB_2656753).

Techniques: Expressing, Membrane, Flow Cytometry, Immunofluorescence, Staining, Fluorescence

Journal: Cancers

Article Title: The Impact of TRAIL on the Immunological Milieu during the Early Stage of Abdominal Sepsis

doi: 10.3390/cancers15061773

Figure Lengend Snippet: Effect of 6h CASP treatment on the expression of TRAIL receptors. TRAIL receptors DR5, DcR1, and DcR2 were stained and assessed by FACS. Representative FACS plots show ( a ) DR5 staining in neutrophils derived from BM and spleen, ( c ) DcR2 staining in neutrophils of the spleen, and ( e ) DcR1 staining in neutrophils of the BM. Quantification of ( b ) DR5, ( d ) DcR2, and ( f ) DcR1 expression in neutrophils of the BM and spleen of n = 5 animals in each group. Neutrophils of septic WT and TRAIL–/– mice were compared with naïve ones. Graphs show mean ± SEM (* p < 0.05, ** p < 0.01).

Article Snippet: Surface staining was performed using antibodies against DcR1 (REA759, Milteny Biotech), DcR2 (mDcR2-1, Biolegend, San Diego, CA, USA), DR5 (REA1233, Milteny Biotech), TRAIL (CD 253) (N2B2, Biolegend), CD45 (30-F11, BD Bioscience), CD172 (P84, eBioscience), CD11b (M1/70, Biolegend), F4/80 (BM8, Biolegend), Ly6G (1A8, Biolegend), and DUMP (live/dead, CD3 (17A2, Biolegend), CD19 + (6D5, Biolegend) and CD49b (HMalpha2, BD Bioscience)).

Techniques: Expressing, Staining, Derivative Assay

Journal: The Journal of Neuroscience

Article Title: Human Astrocytes Are Resistant to Fas Ligand and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis

doi: 10.1523/JNEUROSCI.5572-05.2006

Figure Lengend Snippet: Human astrocytes are resistant to Fas agonist and TRAIL. A, Flow cytometry analysis of the cell surface expression of Fas, DR4, and DR5 in human astrocytes and A549 and U343MG cancer cell lines. B, Western blot detection of DR4 and DR5 expression in human astrocytes, A549, U343MG, and Cos-7 cells transfected with either DR4 or DR5 cDNA. Three antibodies against DR4 and three antibodies to DR5 used in the study are indicated to the left and are described in Materials and Methods. Extracellular signal-regulated kinase 1/2 (ERK1/2) was used as protein loading control. C, RT-PCR analysis of DR4 and DR5 mRNA expression in human astrocytes and control A549 cells. D, Cell viability in human glioma cell line U343MG treated with the various doses of three recombinant forms of human TRAIL, as described in Materials and Methods. E, Cell viability analysis of human astrocytes after exposure to 3 μg/ml Fas agonist monoclonal antibody (Fas mAb), nontagged TRAIL (TRAIL), histidine-tagged TRAIL (his-TRAIL), and antibody cross-linked Flag-tagged TRAIL (Flag-TRAIL) for 24 h. F, Phase-contrast microscopic observation of apoptotic cell death in human glioma cell line U343MG but not human astrocytes after exposure to 3 μg/ml Fas agonist and various forms of recombinant TRAIL. Cell viability data (D, E) were representative from three independent experiments (mean ± SEM; n = 6).

Article Snippet: Cells (10 6 ) were analyzed for the surface expression of DR4 and DR5 by indirect staining with mouse anti-human DR4 and DR5 (Alexis Biochemicals) followed by FITC-conjugated goat anti-mouse IgG antibody (Sigma) ( Song et al., 2003a ).

Techniques: Flow Cytometry, Expressing, Western Blot, Transfection, Reverse Transcription Polymerase Chain Reaction, Recombinant

Journal: The Journal of Neuroscience

Article Title: Human Astrocytes Are Resistant to Fas Ligand and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis

doi: 10.1523/JNEUROSCI.5572-05.2006

Figure Lengend Snippet: Insufficient expression of TRAIL receptors blocks TRAIL-induced signal pathway in human astrocytes. A, B, Analysis of Fas agonist (A) and TRAIL-induced DISC (B) in human astrocytes and human glioma U343MG cells. The DISC was immunoprecipitated by the treatment of human astrocytes with either Fas agonist antibody or Flag-tagged TRAIL for 15 min in stimulated cells (+) or after cell lysis in unstimulated cells (−). Cell lysates obtained from untreated cells (Lysate) were included as control for the expression of the endogenous proteins. C, TRAIL sensitivity of human astrocytes transfected with DR5 cDNA. Human astrocytes were transfected with DR5 cDNA for 48 h and treated with KN93 (100 μm) and TRAIL (1 μg/ml), alone or in combination as indicated at the top. After 6 h of treatment, the cells were subjected to Western blot for DR5 expression (top) and caspase-3 cleavage (middle). After 24 h of treatment, the cells were analyzed for cell viability (bottom) (mean ± SEM; n = 6; *p < 0.05).

Article Snippet: Cells (10 6 ) were analyzed for the surface expression of DR4 and DR5 by indirect staining with mouse anti-human DR4 and DR5 (Alexis Biochemicals) followed by FITC-conjugated goat anti-mouse IgG antibody (Sigma) ( Song et al., 2003a ).

Techniques: Expressing, Immunoprecipitation, Lysis, Transfection, Western Blot

Journal: The Journal of Neuroscience

Article Title: Human Astrocytes Are Resistant to Fas Ligand and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis

doi: 10.1523/JNEUROSCI.5572-05.2006

Figure Lengend Snippet: CaMKII inhibitor KN93 inhibits PEA-15/PED and c-FLIP expression. Human astrocytes were treated or untreated (Medium) with KN93 (100 μm) for 24 h and then subjected to flow cytometry analysis of Fas and DR5 expression (A), Western blot analysis of the expression of key apoptotic proteins (B), and two-dimensional PAGE analysis of PEA-15/PED isoforms (C). The antibodies are indicated to the left (B). The isoelectric direction (H+, OH−) of two-dimensional PAGE is indicated above (C).

Article Snippet: Cells (10 6 ) were analyzed for the surface expression of DR4 and DR5 by indirect staining with mouse anti-human DR4 and DR5 (Alexis Biochemicals) followed by FITC-conjugated goat anti-mouse IgG antibody (Sigma) ( Song et al., 2003a ).

Techniques: Expressing, Flow Cytometry, Western Blot

Journal: The Journal of Neuroscience

Article Title: Human Astrocytes Are Resistant to Fas Ligand and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis

doi: 10.1523/JNEUROSCI.5572-05.2006

Figure Lengend Snippet: Effects of siRNA specific to PEA-15/PED and c-FLIP on Fas and TRAIL of human astrocytes. A, Western blot analysis of the expression of PEA-15/PED and c-FLIP (c-FLIPL, c-FLIPS). Human astrocytes were transfected with PEA-15/PED and c-FLIP siRNA or nonspecific control siRNA (Mock) for 24 h before being subjected to Western blot. ERK1/2 was used as the protein loading control. B, C, PEA-15/PED or c-FLIP siRNA sensitizes human astrocytes to Fas agonist. The astrocytes transfected with siRNA were treated with Fas agonist antibody (1 μg/ml) for 6 h and examined on Western blots (B) and 24 h for cell viability assay (C). D, E, Human astrocytes were nontransfected (first column) or transfected with DR5 cDNA (the second to 10th columns) for 48 h and then either transfected or nontransfected with PEA-15/PED or c-FLIP siRNA for 24 h. The cells were further treated or untreated with nontagged TRAIL (1 μg/ml) for 6 h for Western blot detection of caspase-8 cleavage (D) or for 24 h for cell viability analysis (E) in which each value represents six samples, and * indicates the significance (p < 0.05) (C).

Article Snippet: Cells (10 6 ) were analyzed for the surface expression of DR4 and DR5 by indirect staining with mouse anti-human DR4 and DR5 (Alexis Biochemicals) followed by FITC-conjugated goat anti-mouse IgG antibody (Sigma) ( Song et al., 2003a ).

Techniques: Western Blot, Expressing, Transfection, Viability Assay

Journal: Pharmaceuticals

Article Title: The Synergistic Activity of Bortezomib and TIC10 against A2058 Melanoma Cells

doi: 10.3390/ph14080820

Figure Lengend Snippet: Relative death receptor mRNA expression levels after 72 h treatment with bortezomib (BOZ) or TIC10 and their combinations. The data were normalized to the medium control.

Article Snippet: The cells were stained with 3 μL phycoerythrin (PE) conjugated isotype control (Sony Biotechnology, Weybridge, UK), anti-DR4 antibody (Sony Biotechnology, Weybridge, UK) or anti-DR5 antibody (Sony Biotechnology, Weybridge, UK).

Techniques: Expressing, Control

Journal: Pharmaceuticals

Article Title: The Synergistic Activity of Bortezomib and TIC10 against A2058 Melanoma Cells

doi: 10.3390/ph14080820

Figure Lengend Snippet: Influence on the death receptor 4 (DR4) and death receptor 5 (DR5) expression of melanoma cell line after 72 h exposure. The ratio of the mean fluorescence intensity (RFI) of ( A ) DR4 expression and ( B ) DR5 expression is reported. The data were normalized to the medium control (RFI = treated cells MFI/control cells MFI; MFI: mean fluorescence intensity). Data are presented as mean values ± standard deviation (SD) ( n = 2). The levels of significance are shown as follows: x: p < 0.05; y: p < 0.01; z: p < 0.001, determined by a one-way ANOVA test followed by Fisher’s LSD post hoc test.

Article Snippet: The cells were stained with 3 μL phycoerythrin (PE) conjugated isotype control (Sony Biotechnology, Weybridge, UK), anti-DR4 antibody (Sony Biotechnology, Weybridge, UK) or anti-DR5 antibody (Sony Biotechnology, Weybridge, UK).

Techniques: Expressing, Fluorescence, Control, Standard Deviation