recombinant mouse trail protein  (R&D Systems)

Journal: Nature

Article Title: Glioblastoma-instructed astrocytes suppress tumour-specific T cell immunity

doi: 10.1038/s41586-025-08997-x

Figure Lengend Snippet: a , Pearson correlation of TNFSF10 in the TCGA glioblastoma dataset. b , IL11 expression in a glioblastoma scRNA-seq dataset 4 . OPC, oligodendrocyte progenitor cell; NPC, neural progenitor cell; AC, astrocyte; MES, mesenchymal. c , EGFRvIII immunohistochemistry and IL-11 + glioblastoma cell immunofluorescence. d , Peritumoural IL11RA + TRAIL + astrocyte immunofluorescence in glioblastoma. e , Glioblastoma survival stratified by the first and fourth quartile IL11 expression (data from TCGA). f , IL-11 signalling score (WP2332) in GL261 Aldh1l1 cre-ERT2/TdTomato reporter cell scRNA-seq ( Fig. 2b ). g , IL-11 immunofluorescence in GL261 at 15 days. h , IL-11 concentration in GL261-implanted brain lysates at 15 days (n = 3 each). i , Astrocyte STAT3 phosphorylation after treatment with recombinant mouse IL-11 (rmIL-11) ( n = 3 control, n = 5 TCM). j – l , TNFSF10 expression by quantitative PCR (qPCR) ( j , k ) and flow cytometry ( l ) of primary mouse astrocytes treated with rmIL-11 ( j : n = 23 control, n = 14 rmIL-11, l : n = 3 control, n = 5 rmIL-11) and human astrocytes treated with human IL-11 (rhIL-11) ( k ; n = 5 control, n = 11 rhIL-11). m – o , TNFSF10 expression by qPCR ( m , n ) and flow cytometry ( o ) in IL-11-treated mouse ( m , n = 4 each) and human ( n , n = 5 each) astrocytes with STAT3 inhibition. p , q , TNFSF10 expression in GL261 TCM-treated mouse ( p ) and U87 TCM-treated human ( q ) astrocytes and IL-11 neutralization ( n = 5 each). r , GL261 survival after astrocyte Il11ra1 inactivation ( n = 18 NT sgRNA, n = 28 sgIl11ra1). Weight fold change in survivors, sgIl11ra1 1.064 ± 0.028 g, NT sgRNA 1.035 ± 0 g (sgIl11ra1 versus NT sgRNA, P = 0.7015 two-tailed t -test). s , t , TRAIL + ( s ) and pSTAT3 + TRAIL + ( t ) TdTomato + reporter cells in GL261 at 15 days ( n = 3 NT sgRNA, n = 5 sgIl11ra1). u , Apoptotic CD4 + and CD8 + tumour-infiltrating T cells ( n = 5 each). v , Survival in IL11 -overexpressing GL261 mice ( n = 10 each). w–y , TRAIL + ( w ) and pSTAT3 + TRAIL + TdTomato + reporter cells ( x ), and apoptotic T cells ( y ) in IL11 -overexpressing GL261 mice at 12 days ( n = 5 each). Data are the mean ± s.e.m. Unpaired two-tailed t -test. log-rank (Mantel–Cox) test ( e , r , v ). n indicates biologically independent samples. Scale bars, 25 μm ( d , right) 50 μm ( c , right; g , bottom; d , left bottom), 100 μm ( c , left; d , left top), 500 μm ( g , top).

Article Snippet: To measure nuclear translocation of NFAT2, naive splenic CD4 T cells were isolated by magnetic selection as described above and activated with 10 μg ml −1 plate-bound anti-CD3 and 0.25 μg ml −1 soluble anti-CD28, with or without 10 μg ml −1 plate-bound recombinant TRAIL (R&D Systems, 1121-TL-010) in complete RPMI for 30 min. T cells were then stained with FITC anti-CD3 (1:100, Thermo Fisher Scientific, 11–0032-82), followed by fixation and permeabilization with eBioscience FOXP3/Transcription Factor Staining buffer set (Thermo Fisher Scientific, 00–5523-00) following the manufacturer’s instructions and intracellular staining with PE NFATc1 (1:50, BioLegend, 649606) and DAPI (Sigma-Aldrich, D9542) for 1 h. Around 5,000 cells of each condition were measured on an Imagestream X Mark II (Cytek Biosciences), and the Similarity function in IDEAS software (Amnis, v.6.2) was used to calculate the overlap between DAPI and NFAT2 signals.

Techniques: Derivative Assay, Expressing, Immunohistochemistry, Immunofluorescence, Concentration Assay, Phospho-proteomics, Recombinant, Control, Real-time Polymerase Chain Reaction, Flow Cytometry, Inhibition, Neutralization, Two Tailed Test

Journal: Nature

Article Title: Glioblastoma-instructed astrocytes suppress tumour-specific T cell immunity

doi: 10.1038/s41586-025-08997-x

Figure Lengend Snippet: ( a,b ) Activation/exhaustion profiling by flow cytometry of tumour-infiltrating PD-1 + CD8 + (a) or PD-1 + CD4 + (b) T cells following genetic perturbation of TRAIL in astrocytes (n = 7 NTsgRNA, n = 9 sg Tnfsf10 ). ( c ) Flow cytometry analysis of T-cell exhaustion markers in tumour-infiltrating CD8 + (left) or CD4 + (right) T cells following genetic perturbation of TRAIL in astrocytes (n = 7 NTsgRNA, n = 9 sg Tnfsf10 ). ( d ) Tox expression measured by qPCR in GL261-infiltrating CD4 or CD8 T cells following genetic inactivation of TRAIL in astrocytes. ( e,f ) Flow cytometry analysis and representative plots of cytokines after restimulation with PMA/Ionomycin in tumour-infiltrating CD8 + (e) or CD4 + (f) T cells following genetic perturbation of TRAIL in astrocytes (n = 7 NTsgRNA, n = 9 sg Tnfsf10 ). ( g ) Nuclear translocation of NFAT2 analysed by imaging flow cytometry in splenic CD4 + T cells activated in vitro with anti-CD3/CD28 alone or in combination with recombinant TRAIL as indicated. Violin plot of similarity score between DAPI and NFAT2 analysed using the Mann-Whitney U-test with Sidak’s correction (left) and representative images (right) are shown. ( h ) DR5 expression median fluorescence intensity (MFI) and histogram distribution in T cell and myeloid compartments from GL261-bearing mice following genetic perturbation with control NTsgRNA (n = 7 mice). ( i ) Percentage of apoptotic microglia and monocyte-derived macrophages (MDM) in GL261-bearing mice 15 days following genetic inactivation of TRAIL in astrocytes. ( j,k ) Differential expression analysis by bulk RNA-seq in microglia (j) and flow cytometry analysis of microglia and MDM isolated from GL261-bearing Rag2−/− mice undergoing sg Tnfsf10 or NTsgRNA genetic perturbation of astrocytes (k). ( l ) DR5 median fluorescence intensity and histogram in GL261-infiltrating dendritic cell subtypes. ( m ) Purity analysis of mixed glia culture, using GFAP, CD45, CD11b, O4, Ter119 as lineage markers. Data shown as mean ± SEM. Unpaired two-tailed t-test. n indicates biologically independent samples.

Article Snippet: To measure nuclear translocation of NFAT2, naive splenic CD4 T cells were isolated by magnetic selection as described above and activated with 10 μg ml −1 plate-bound anti-CD3 and 0.25 μg ml −1 soluble anti-CD28, with or without 10 μg ml −1 plate-bound recombinant TRAIL (R&D Systems, 1121-TL-010) in complete RPMI for 30 min. T cells were then stained with FITC anti-CD3 (1:100, Thermo Fisher Scientific, 11–0032-82), followed by fixation and permeabilization with eBioscience FOXP3/Transcription Factor Staining buffer set (Thermo Fisher Scientific, 00–5523-00) following the manufacturer’s instructions and intracellular staining with PE NFATc1 (1:50, BioLegend, 649606) and DAPI (Sigma-Aldrich, D9542) for 1 h. Around 5,000 cells of each condition were measured on an Imagestream X Mark II (Cytek Biosciences), and the Similarity function in IDEAS software (Amnis, v.6.2) was used to calculate the overlap between DAPI and NFAT2 signals.

Techniques: Activation Assay, Knockdown, Flow Cytometry, Expressing, Translocation Assay, Imaging, In Vitro, Recombinant, MANN-WHITNEY, Fluorescence, Control, Derivative Assay, Quantitative Proteomics, RNA Sequencing, Isolation, Two Tailed Test

Journal: Nature

Article Title: Glioblastoma-instructed astrocytes suppress tumour-specific T cell immunity

doi: 10.1038/s41586-025-08997-x

Figure Lengend Snippet: ( a,b ) IL11 expression per cell type (a) and GBM subtype (b) in TCGA after cell-type deconvolution. ( c ) IL11 (left) and IL11RA expression by cell type in scRNA-seq 22 . ( d ) Spatially annotated bulk RNA-seq from Ivy Glioblastoma Atlas showing expression correlation of STAT3 signalling program (M5897) and TRAIL-driven apoptosis signalling (M23448) split by anatomical structure. ( e ) Spatial transcriptomics analysis of human GBM performed with SPATA2 86 showing spatially-weighted correlation of IL11 expression with GBM subtype metaprograms 32 . ( f ) IL11RA expression by time to recurrence in GBM-associated astrocytes analysed by scRNA-seq in Fig. 1b . ( g ) IL11RA expression in GBM-associated astrocytes determined by scRNA-seq. ( h ) Il11ra1 expression in GL261-associated astrocytes determined by scRNA-seq. ( i-k ) IL-11 quantified by ELISA in conditioned media from primary human GBM lines (i, n = 4 each), primary mouse astrocytes or GL261 cultures (j, n = 3 astrocytes, n = 6 GL261), or genetically-engineered glioma models (k, n = 4 each). ( l ) pSTAT3 (Y705) in primary murine astrocytes stimulated with rmIL-11 for 15 mins. ( m ) TNFSF10 promoter luciferase reporter activity in HepG2 cells treated with rhIL-11 for 24 or 48 h. ( n ) Tnfsf10 expression measured by qPCR in purified cultures of either microglia, oligodendrocytes, or neurons overnight with recombinant IL-11. ( o ) IL15 and TGFB1 expression in GBM cells analysed by scRNA-seq 4 . ( p ) Tnfsf10 expression measured by qPCR in primary mouse astrocyte cultures stimulated with the indicated cytokines. Data shown as mean ± SEM. Unpaired two-tailed t-test and two-way ANOVA used. n indicates biologically independent samples.

Article Snippet: To measure nuclear translocation of NFAT2, naive splenic CD4 T cells were isolated by magnetic selection as described above and activated with 10 μg ml −1 plate-bound anti-CD3 and 0.25 μg ml −1 soluble anti-CD28, with or without 10 μg ml −1 plate-bound recombinant TRAIL (R&D Systems, 1121-TL-010) in complete RPMI for 30 min. T cells were then stained with FITC anti-CD3 (1:100, Thermo Fisher Scientific, 11–0032-82), followed by fixation and permeabilization with eBioscience FOXP3/Transcription Factor Staining buffer set (Thermo Fisher Scientific, 00–5523-00) following the manufacturer’s instructions and intracellular staining with PE NFATc1 (1:50, BioLegend, 649606) and DAPI (Sigma-Aldrich, D9542) for 1 h. Around 5,000 cells of each condition were measured on an Imagestream X Mark II (Cytek Biosciences), and the Similarity function in IDEAS software (Amnis, v.6.2) was used to calculate the overlap between DAPI and NFAT2 signals.

Techniques: Expressing, RNA Sequencing, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Purification, Recombinant, Two Tailed Test

Journal: Nature

Article Title: Glioblastoma-instructed astrocytes suppress tumour-specific T cell immunity

doi: 10.1038/s41586-025-08997-x

Figure Lengend Snippet: ( a ) Cleaved caspase-3/7 + and cleaved caspase-8 + CD4/8 + T cells in co-cultures with GL261 TCM pre-treated astrocytes with IL-11 neutralization (n = 6 CD4 + groups; n = 3 CD8 + anti-Isotype; n = 4 CD8 + anti-IL-11). ( b ) Cleaved caspase-3/7 + and cleaved caspase-8 + CD4/8 + T cells co-cultured with rmIL-11-treated astrocytes (n = 11 CD4 + control; n = 6 CD4 + rmIL-11; n = 7 CD8 + control; n = 4 CD8 + rmIL-11). ( c ) Percentage of cleaved caspase-3/7 + (left) and cleaved caspase-8 + (right) CD4 + or CD8 + T cells co-cultured with rmIL-11 pre-treated astrocytes and TRAIL-blocking antibodies (n = 6 CD4 + ; n = 3 CD8 + anti-Isotype; n = 4 CD8 + anti-TRAIL). ( d ) Percentage of cleaved caspase-3/7 + CD4 + T cells co-cultured with astrocytes pre-treated with rmIL-11 in combination with STAT3 inhibitor or vehicle. TRAIL-blocking or control antibodies, or recombinant TRAIL were added at start of co-culture. (n = 6 each). ( e ) Validation of astrocyte-specific Il11ra1 knockdown by immunofluorescence. ( f ) Absolute cell count of the indicated compartments in GL261-bearing mice following astrocyte-specific Il11ra1 or non-targeting control inactivation. ( g,h ) IL-11 concentration in TCM (g) (n = 4 empty vector; n = 2 IL-11 OE) and growth dynamics (h) (n = 6 empty vector; n = 2 IL-11 OE) of empty vector transfected or IL-11 overexpressing (OE) GL261 cells (n = 3 each). ( i ) Relative increase in bioluminescence tumour size between days 6 and 11 in control or IL-11-overexpressing tumours. ( j ) Analysis of the Il11 promoter showing multiple AHR consensus binding sites. ( k,l ) Expression of Il11 (left) and Cyp1b1 (right) in GL261 cells harboring control- or AHR-deletion (k) or a constitutively activated form of AHR (l). ( m,n ) Mouse GL261 (m) or human U87 (n) GBM cells stimulated with L-Kynurenine (Kyn) alone or in combination with AHR inhibitor CH223191 (AHRi). Data shown as mean ± SEM. Unpaired two-tailed t-test and two-way ANOVA used. n indicates biologically independent samples.

Article Snippet: To measure nuclear translocation of NFAT2, naive splenic CD4 T cells were isolated by magnetic selection as described above and activated with 10 μg ml −1 plate-bound anti-CD3 and 0.25 μg ml −1 soluble anti-CD28, with or without 10 μg ml −1 plate-bound recombinant TRAIL (R&D Systems, 1121-TL-010) in complete RPMI for 30 min. T cells were then stained with FITC anti-CD3 (1:100, Thermo Fisher Scientific, 11–0032-82), followed by fixation and permeabilization with eBioscience FOXP3/Transcription Factor Staining buffer set (Thermo Fisher Scientific, 00–5523-00) following the manufacturer’s instructions and intracellular staining with PE NFATc1 (1:50, BioLegend, 649606) and DAPI (Sigma-Aldrich, D9542) for 1 h. Around 5,000 cells of each condition were measured on an Imagestream X Mark II (Cytek Biosciences), and the Similarity function in IDEAS software (Amnis, v.6.2) was used to calculate the overlap between DAPI and NFAT2 signals.

Techniques: Expressing, Neutralization, Cell Culture, Control, Blocking Assay, Recombinant, Co-Culture Assay, Biomarker Discovery, Knockdown, Immunofluorescence, Cell Counting, Concentration Assay, Plasmid Preparation, Transfection, Binding Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Heparan sulfate promotes TRAIL-induced tumor cell apoptosis

doi: 10.1101/2023.07.26.550758

Figure Lengend Snippet: (A) The sensitivity of U266 and IM-9 cells to TRAIL (10, 30, 100, 300ng/ml) was determined by Annexin V-FITC assay. (B) TRAIL (100 ng/ml)-induced apoptosis of IM-9 and U266 cells, with or without HL-III pretreatment, were determined by Annexin V-FITC assay. (C) Expression of TRAIL receptor DR4 and DR5 on U266B1, RPMI-8226 and IM-9 cells were determined with PE conjugated mAbs against DR4 and DR5 using FACS. The shaded histograms are from cells stained with mouse IgG1-PE conjugate. (D) Expressions of cell surface HS on untreated cells or cells pretreated with HL-III were determined by a human anti-HS mAb, followed by an anti-human-IgG Alexa-594 secondary antibody. The shaded histogram is from cells stained with secondary antibody only. (E) Expression of cell surface syndecan-1 was determined by a mouse anti- syndecan-1 mAb followed by an anti-mouse-IgG Alexa-488 secondary antibody. The shaded histogram is from cells stained with secondary antibody only. Error bars represent S.D. ** represents p < 0.01 and *** represents p < 0.0001 by Student’s t test. Data are representative of at least three separate assays.

Article Snippet: Recombinant mouse DR5 (aa53-177)-Fc fusion protein (721-DR, R&D) alone (10μg), or DR5– mTRAIL complex (10 μg each pre-incubated for 1 h at room temperature), were loaded onto heparin-Sepharose (Cytiva) gravity column (200 μl bed volume).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: Heparan sulfate promotes TRAIL-induced tumor cell apoptosis

doi: 10.1101/2023.07.26.550758

Figure Lengend Snippet: (A) While DR5 does not bind heparin by itself (left half of the gel), DR5-TRAIL complex can bind heparin (right half of the gel), indicating DR5-TRAIL-heparin can form a ternary complex through TRAIL. Representative of three experiments with identical results (B) DR5 and HS bind to different surfaces on TRAIL. Crystal structure of hTRAIL-DR5 complex (IDU3). hTRAIL is shown in cartoon and the three monomers are displayed in green, salmon and gold, respectively. The three DR5 molecules are shown in gray cartoon. Because residues 114- 119 of hTRAIL are disordered in this structure, these residues ( 114 VRERGP 119 , backbone shown in gray random coils) were manually modeled onto the last visible N-terminal residue (Q120) of the hTRAIL. Sidechains responsible for HS binding (from R115, R117 and R121) are shown in sticks. (C-E) TRAIL-dependent internalization of DR4 and DR5 was determined by a FACS-based assay. Cell surface levels of TRAIL receptor DR5(C) and DR4 (D) were determined before TRAIL stimulation, and 30min and 1h after TRAIL stimulation. The shaded histograms are from cells stained mouse IgG1-PE conjugate. (E) Plot of time-dependent internalization of cell surface DR5, with or without HL-III treatment. n = 3. Data is representative of three experiments with similar results.

Article Snippet: Recombinant mouse DR5 (aa53-177)-Fc fusion protein (721-DR, R&D) alone (10μg), or DR5– mTRAIL complex (10 μg each pre-incubated for 1 h at room temperature), were loaded onto heparin-Sepharose (Cytiva) gravity column (200 μl bed volume).

Techniques: Residue, Binding Assay, Staining

Journal: Journal of cellular physiology

Article Title: Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

doi: 10.1002/jcp.30863

Figure Lengend Snippet: FIGURE 1 Involvement of Bid (BH3‐interacting domain death agonist) in the enhancement of cytotoxicity during treatment of artesunate (ART) and tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL). HCT116 wild‐type (WT) and Bid knockout (KO) cells were pretreated with artesunate (ART, 50 μM) for 20 h and then exposed to human recombinant TRAIL (2ng/ml) for an additional 4 h. (a) Phase‐contrast images were visualized under a bright‐field microscope. Representative images are shown (scale bar: 100 µm). (b) Cell death was determined using a trypan blue exclusion assay. Error bars represent the mean± SD from triplicate experiments. For statistical analysis, two‐way analsis of variance followed by Tukey's post hoc test was used. ***p < 0.001. (c) For fluorescence images, cells were stained with propidium iodide (PI) and Hoechst 33342. Representative images are shown (scale bar: 25 µm).

Article Snippet: Mouse recombinant TRAIL was purchased from R&D Systems.

Techniques: Knock-Out, Recombinant, Microscopy, Trypan Blue Exclusion Assay, Fluorescence, Staining

Journal: Journal of cellular physiology

Article Title: Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

doi: 10.1002/jcp.30863

Figure Lengend Snippet: FIGURE 2 Role of Bid (BH3‐interacting domain death agonist) in the synergistic interaction between artesunate/erastin (ART/ERA) and tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) in HCT116 cells. HCT116 wild‐type (WT) and Bid knockout (KO) cells were pretreated with ART (50 μM) for 20 h and then exposed to human recombinant TRAIL (2 ng/ml) for an additional 4 h. (a) Whole‐cell extracts were analyzed with an immunoblotting assay using indicated antibodies. (b) Densitometry analysis of the bands from the western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, two‐way analysis of variance (ANOVA), followed by Tukey's post hoc test was used. ***p < 0.001 versus the Bid KO group. HCT116 WT cells were pretreated with ERA (50 μM) for 20 h and then exposed to human recombinant TRAIL (2 ng/ml) for an additional 4 h. (c) Whole‐cell extracts were analyzed with the immunoblotting assay using indicated antibodies. (d) Densitometry analysis of the bands from the western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, one‐way ANOVA was used. ***p < 0.001 versus the MOCK group. (e) Alteration of mitochondrial membrane potential was detected using JC‐1 dye after treatment. Red and green fluorescence represents the aggregate and monomeric form of JC‐1, respectively (scale bar: 50 μm). PARP, poly (ADP‐ribose) polymerase; tBid, truncated Bid.

Article Snippet: Mouse recombinant TRAIL was purchased from R&D Systems.

Techniques: Knock-Out, Recombinant, Western Blot, Membrane, Fluorescence

Journal: Journal of cellular physiology

Article Title: Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

doi: 10.1002/jcp.30863

Figure Lengend Snippet: FIGURE 4 Role of caspase‐8 in the synergistic interaction between artesunate (ART) and tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL). HCT116 wild‐type (WT) cells were pretreated with ART (50 μM) for 20 h and then exposed to human recombinant TRAIL (2 ng/ml) for an additional 4 h. Caspase‐8 inhibitor Z‐IETD‐FMK (20 μM) was added 2 h before TRAIL treatment. (a) Cell death was determined using a trypan blue exclusion assay. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, one‐way analysis of variance (ANOVA) was used. ***p < 0.001. (b) Whole‐cell lysates were analyzed with an immunoblotting assay using indicated antibodies. (c) Densitometry analysis of bands in western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, one‐way ANOVA was used. ***p < 0.001. (d) Caspase‐8 activity was measured as described in Materials and Methods section. All experiments were performed in triplicates and caspase‐8 activity was plotted as average relative fluorescence units (RFUs). PARP, poly (ADP‐ribose) polymerase; tBid, truncated Bid.

Article Snippet: Mouse recombinant TRAIL was purchased from R&D Systems.

Techniques: Recombinant, Trypan Blue Exclusion Assay, Western Blot, Activity Assay, Fluorescence

Journal: Journal of cellular physiology

Article Title: Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

doi: 10.1002/jcp.30863

Figure Lengend Snippet: FIGURE 5 Employment of Bid (BH3‐interacting domain death agonist) mutants for investigating the role of Bid in the synergistic apoptosis. HCT116 wild‐type (WT) or Bid knockout (KO) cells stably expressing control vector, Bid, Bid D60E, or Bid G94E were pretreated with artesunate (ART) (50 μM) for 20 h and then exposed to human recombinant tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) (2 ng/ml) for an additional 4 h. (a) Cell death was determined using trypan blue exclusion assay. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, two‐way analysis of variance (ANOVA), followed by Tukey's post hoc test was used. ***p < 0.001. (b, d) Cell lysates were analyzed with an immunoblotting assay using indicated antibodies. (c, e) Densitometry analysis of bands in western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, one‐way ANOVA was used. **p < 0.01, ***p < 0.001 versus the vector group in (c). ***p < 0.001 versus the D60E group, ###p < 0.001 versus the G94E group in (e). PARP, poly (ADP‐ribose) polymerase; tBid, truncated Bid.

Article Snippet: Mouse recombinant TRAIL was purchased from R&D Systems.

Techniques: Knock-Out, Stable Transfection, Expressing, Control, Plasmid Preparation, Recombinant, Trypan Blue Exclusion Assay, Western Blot

Journal: Journal of cellular physiology

Article Title: Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

doi: 10.1002/jcp.30863

Figure Lengend Snippet: FIGURE 6 Translocation of truncated Bid (BH3‐interacting domain death agonist) to mitochondria during combined treatment of artesunate (ART) and tumor necrosis factor‐related apoptosis‐ inducing ligand (TRAIL). HCT116 wild‐type (WT) or Bid knockout (KO) cells stably expressing control vector, Bid, Bid D60E, or Bid G94E were pretreated with ART (50 μM) for 20 h and then exposed to human recombinant TRAIL (2 ng/ml) for an additional 4 h. After treatments, mitochondrial and cytosol fractions were isolated as described in the Materials and Methods section. Each fraction was analyzed with an immunoblotting assay using indicated antibodies. tBid, truncated Bid.

Article Snippet: Mouse recombinant TRAIL was purchased from R&D Systems.

Techniques: Translocation Assay, Knock-Out, Stable Transfection, Expressing, Control, Plasmid Preparation, Recombinant, Isolation, Western Blot

Journal: Journal of cellular physiology

Article Title: Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

doi: 10.1002/jcp.30863

Figure Lengend Snippet: FIGURE 7 Artesunate (ART)‐induced endoplasmic reticulum stress response and death receptor 5 (DR5) expression. HCT116 wild‐type (WT) or Bid (BH3‐interacting domain death agonist) knockout (KO) cells stably expressing control vector, Bid, Bid D60E, or Bid G94E were pretreated with ART (50 μM) for 20 h and then exposed to human recombinant tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) (2 ng/ml) for an additional 4 h. Z‐IETD‐FMK (20 μM) was added 2 h before TRAIL treatment. (a–d) Cell lysates were analyzed with an immunoblotting assay using indicated antibodies. Densitometry analysis of bands in western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, one‐way analysis of variance was used. ***p < 0.001 versus the MOCK group.

Article Snippet: Mouse recombinant TRAIL was purchased from R&D Systems.

Techniques: Expressing, Knock-Out, Stable Transfection, Control, Plasmid Preparation, Recombinant, Western Blot

Journal: Journal of cellular physiology

Article Title: Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

doi: 10.1002/jcp.30863

Figure Lengend Snippet: FIGURE 8 Truncation of Bid (BH3‐interacting domain death agonist) in CHOP (CCAAT‐enhancer‐binding protein homologous protein)‐ deficient cells. mouse embryonic fibroblast (MEF) wild‐type (WT) and CHOP knockout (KO) cells were pretreated with artesunate (ART) (50 μM) for 20 h and then exposed to mouse recombinant tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) (100 ng/ml) for an additional 4 h. (a) Whole‐cell extracts were analyzed with an immunoblotting assay using indicated antibodies. (b) Densitometry analysis of the bands from the western blot was performed. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, two‐way analysis of variance, followed by Tukey's post hoc test was used. ***p < 0.001 versus the CHOP KO group. PARP, poly (ADP‐ribose) polymerase; tBid, truncated Bid.

Article Snippet: Mouse recombinant TRAIL was purchased from R&D Systems.

Techniques: Binding Assay, Knock-Out, Recombinant, Western Blot