Journal:

Article Title: Requirement of p53 targets in chemosensitization of colonic carcinoma to death ligand therapy

doi: 10.1073/pnas.2435285100

Figure Lengend Snippet: The effects of KILLER/DR5, Bak inhibition, and caspase 8 or 9 inhibition on sensitization of Bax-/- cells to TRAIL after DNA damage. Bax-/- HCT116 cells were transfected with KILLER/DR5 siRNA (A) or Bak siRNA (B). LacZ siRNA oligos were used as a control. Twenty-four hours after transfection, cells were treated as described in the legend to Fig. 1. KILLER/DR5 and Bak expression were determined by Northern blots. Cells were harvested, stained with propidium iodide, and analyzed by flow cytometry. The sub-G1 content of 20,000 cells was examined for each sample. (C) Differential effects of caspase 8 (Z-IETD-FMK) and caspase 9 (Z-LEHD-FMK) inhibitors on TRAIL-induced apoptosis. Wild-type or Bax-/- HCT116 cells were pretreated with 10 μg/ml CPT-11 for 16 h, after which 20 μM caspase 8 or 9 inhibitor was added; after 2 h, TRAIL (10 ng/ml) plus 1 μg/ml anti-His-6 was added for 4 h before sub-G1 analysis. Extracts were collected and analyzed by Western blotting for the expression of caspase 9.

Article Snippet: Polyclonal antibody against caspase 9 was purchased from Imgenex (San Diego).

Techniques: Inhibition, Transfection, Expressing, Northern Blot, Staining, Flow Cytometry, Western Blot

Journal: Frontiers in Immunology

Article Title: Immune biomarkers in circulating cells of NSCLC patients can effectively evaluate the efficacy of chemotherapy combined with anti-PD-1 therapy

doi: 10.3389/fimmu.2025.1521708

Figure Lengend Snippet: T-cell subpopulations were determined via flow cytometry. (A) Gating strategy for aβT cell subpopulations in flow cytometry: Gate CD3+ T cells, CD3+CD4+ T cells, CD3+CD8+ T cells, CD3+CD4-CD8- T cells, CD3+PD-1+ T cells, CD3+TIM-3+ T cells, CD3+TIM-3+PD-1+ T cells, CD3+CD4+PD-1+ T cells, CD3+CD4+TIM-3+ T cells, CD3+CD4+TIM-3+PD-1+ T cells, CD3+CD4+CD28+ T cells, CD3+CD4+CD28- T cells, CD3+CD8+PD-1+ T cells, CD3+CD8+TIM-3+ T cells, CD3+CD8+TIM-3+PD-1+ T cells, CD3+CD8+CD28+ T cells, CD3+CD8+CD28- T cells, separately. (B) Gating strategy for γδT cell subpopulations in flow cytometry: Gate CD3+γδT+ T cells, CD3+γδT+V81+ T cells, CD3+γδT+Vδ2+ T cells, CD3+γδT+PD-1+ T cells, CD3+γδT+TIM-3+ T cells, CD3+γδT+TIM-3+PD-1+ T cells, CD3+γδT+CD28+ T cells, CD3+γδT+CD28- T cells, CD3+γδT+Vδ1+PD-1+ T cells, CD3+γδT+Vδ1+TIM-3+ T cells, CD3+γδT+Vδ1+TIM-3+PD-1+ T cells, CD3+γδT+Vδ1+CD28+ T cells, CD3+γδT+Vδ1+CD28- T cells, CD3+γδT+Vδ2+PD-1+ T cells, CD3+γδT+Vδ2+TIM-3+ T cells, CD3+γδT+Vδ2+TIM-3+PD-1+ T cells, CD3+γδT+Vδ2+CD28+ T cells, CD3+γδT+Vδ2+CD28- T cells, separately.

Article Snippet: Panel 1 included PerCP-Vio700 anti-human Vδ1 (clone: REA173, cat No: 130-120-441, Miltenyi), phycoerythrin (PE) anti-human Vδ2 (clone: B6, cat No: 331408, Biolegend), APC-H7 anti-human CD3 (clone: SK7, cat No: 560176, BD Biosciences), PE-Cy7 anti-human CD28 (clone: CD28.2, cat No: 302926, Biolegend), R718 anti-human γδ TCR (clone: 11F2, cat No: 752023, BD Biosciences), FITC anti-human CD279 (programmed cell death protein 1, PD-1) (clone: EH12.2H7, cat No: 329904, Biolegend), Alexa Fluor 647 anti-human CD366 (T-cell immunoglobulin domain and mucin domain-3, TIM-3) (clone: 7D3, cat No: 565558, BD Biosciences); Panel 2 included Alexa Fluor 700 anti-human CD3 (clone: OKT3, cat No: 317340, Biolegend), APC-Cy7 anti-human CD4 (clone: RPA-T4, cat No: 557871, BD Biosciences), PerCP-Cy5.5 anti-human CD8 (clone: SK1, cat No: 565310, BD Biosciences), PE-Cy7 anti-human CD28 (clone: CD28.2, cat No: 302926, Biolegend), FITC anti-human CD279 (programmed cell death protein 1, PD-1) (clone: EH12.2H7, cat No: 329904, Biolegend), and Alexa Fluor 647 anti-human CD366 (T cell immunoglobulin domain and mucin domain-3, TIM-3) (clone: 7D3, cat No: 565558, BD Biosciences).

Techniques: Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Immune biomarkers in circulating cells of NSCLC patients can effectively evaluate the efficacy of chemotherapy combined with anti-PD-1 therapy

doi: 10.3389/fimmu.2025.1521708

Figure Lengend Snippet: Characteristics of baseline lymphocyte subpopulations and cytokines.

Article Snippet: Panel 1 included PerCP-Vio700 anti-human Vδ1 (clone: REA173, cat No: 130-120-441, Miltenyi), phycoerythrin (PE) anti-human Vδ2 (clone: B6, cat No: 331408, Biolegend), APC-H7 anti-human CD3 (clone: SK7, cat No: 560176, BD Biosciences), PE-Cy7 anti-human CD28 (clone: CD28.2, cat No: 302926, Biolegend), R718 anti-human γδ TCR (clone: 11F2, cat No: 752023, BD Biosciences), FITC anti-human CD279 (programmed cell death protein 1, PD-1) (clone: EH12.2H7, cat No: 329904, Biolegend), Alexa Fluor 647 anti-human CD366 (T-cell immunoglobulin domain and mucin domain-3, TIM-3) (clone: 7D3, cat No: 565558, BD Biosciences); Panel 2 included Alexa Fluor 700 anti-human CD3 (clone: OKT3, cat No: 317340, Biolegend), APC-Cy7 anti-human CD4 (clone: RPA-T4, cat No: 557871, BD Biosciences), PerCP-Cy5.5 anti-human CD8 (clone: SK1, cat No: 565310, BD Biosciences), PE-Cy7 anti-human CD28 (clone: CD28.2, cat No: 302926, Biolegend), FITC anti-human CD279 (programmed cell death protein 1, PD-1) (clone: EH12.2H7, cat No: 329904, Biolegend), and Alexa Fluor 647 anti-human CD366 (T cell immunoglobulin domain and mucin domain-3, TIM-3) (clone: 7D3, cat No: 565558, BD Biosciences).

Techniques:

Journal:

Article Title: Detection of Direct Binding of Human Herpesvirus 8-Encoded Interleukin-6 (vIL-6) to both gp130 and IL-6 Receptor (IL-6R) and Identification of Amino Acid Residues of vIL-6 Important for IL-6R-Dependent and -Independent Signaling

doi: 10.1128/JVI.75.7.3325-3334.2001

Figure Lengend Snippet: Diagrammatic representations of the cytokine receptor homology and immunoglobulin-like domains (CHD and Ig, respectively) of gp130 and IL-6R, their interactions with hIL-6, and complex formation resulting from these interactions. The figure on the left is a side view that indicates the general locations (asterisks) of receptor residues known to interact with hIL-6. Sites II and III of hIL-6 interact with distinct gp130 molecules to form, in association with IL-6R, the functional hexameric complex, as shown schematically in the right panel (top view); for IL-6R, only the hIL-6 site I-interacting CHD region is depicted (individual, lightly shaded circle), with CHD (site II-interacting) and Ig (site III-interacting) regions of gp130 indicated as adjoined lightly and darkly shaded circles, respectively. There is strong experimental evidence for an IL-6R–gp130 dimerization interface (27, 37), here indicated by double-headed arrows.

Article Snippet: Analogous experiments were undertaken with rhIL-6 (R&D Systems catalog no. 206-IL) using 100 ng of the cytokine per binding assay, and immunological detection was achieved using HRP-conjugated α-hIL-6 antibody (R&D Systems catalog no. AB-206-NA).

Techniques: Functional Assay

Journal:

Article Title: Detection of Direct Binding of Human Herpesvirus 8-Encoded Interleukin-6 (vIL-6) to both gp130 and IL-6 Receptor (IL-6R) and Identification of Amino Acid Residues of vIL-6 Important for IL-6R-Dependent and -Independent Signaling

doi: 10.1128/JVI.75.7.3325-3334.2001

Figure Lengend Snippet: In vitro binding of vIL-6 to IL-6R and gp130. (A) ELISA techniques (see Materials and Methods) were used for the detection of binding by vIL-6 and hIL-6 of sgp130 and sIL-6R. For vIL-6, various amounts (25 to 200 ng) of bacterially produced, purified His6–vIL-6 fusion protein were applied to microassay plate wells coated with sIL-6R or sgp130 or to untreated wells (Blk). After incubation and washing, bound ligand was detected with vIL-6 rabbit antiserum (35) and HRP-conjugated α-rabbit-IgG secondary antibody. Visualization and quantitation was carried out using ABTS reagent and the determination of the OD450 (top panel). Analogous assays were undertaken with rhIL-6 using HRP-conjugated α-hIL-6 as the detection antibody. Experiments were performed in triplicate using 100 ng of ligand (bottom panel). (B) vIL-6 binding to IL-6R and gp130 was determined also by coprecipitation assays using transfected cell media containing sIL-6R–Fc (RFc) or sgp130-Fc (gpFc) together with media containing vIL-6 (see Materials and Methods). Protein A-Sepharose-precipitated material was analyzed by Western blot for the detection of vIL-6. Analogous experiments were undertaken with hIL-6. Inclusion of sgp130 in the sIL-6R–Fc/ligand-binding assays, followed by Western analysis to detect coprecipitated sgp130, demonstrated that vIL-6, in addition to hIL-6, could induce gp130–IL-6R complexing. Experiments using vIL-6 or hIL-6 in the absence of Fc fusion protein or sIL-6R-Fc plus sgp130 in the absence of ligand were included to control for nonspecific binding of proteins to protein A-Sepharose.

Article Snippet: Analogous experiments were undertaken with rhIL-6 (R&D Systems catalog no. 206-IL) using 100 ng of the cytokine per binding assay, and immunological detection was achieved using HRP-conjugated α-hIL-6 antibody (R&D Systems catalog no. AB-206-NA).

Techniques: In Vitro, Binding Assay, Enzyme-linked Immunosorbent Assay, Produced, Purification, Incubation, Quantitation Assay, Transfection, Western Blot, Ligand Binding Assay