Journal: Oncology Reports

Article Title: Expression level of Bcl-XL critically affects sensitivity of hepatocellular carcinoma cells to LIGHT-enhanced and interferon-γ-induced apoptosis

doi: 10.3892/or.17.5.1067

Figure Lengend Snippet: Figure 3. Expression levels of caspase-3, caspase-8, caspase-9, Bcl-XL, Bak and Bid in Hep3B cells treated with IFN-Á/LIGHT. (A) Hep3B cells were treated with 100 ng/ml IFN-Á and various concentrations of LIGHT for 24 h or 72 h as indicated. Cell lysates containing 30 μg of protein were subjected to 12% Tris-glycine gel electrophoresis followed by Western blotting with rabbit polyclonal antibodies against caspase-3, caspase-8, caspase-9, Bcl- XL, Bak, Bid, respectively. Cells treated with 100 ng/ml IFN-Á or 100 ng/ml LIGHT alone and untreated cells were used as the controls. ß-actin was used as an internal control for total protein loading. (B) Hep3B cells (2x105) treated with 100 ng/ml sLIGHT and 100 ng/ml of IFN-Á were also incubated with 100 μM of caspase-3 inhibitor Z-DEVD-fmk, caspase-9 inhibitor Z- IEHD-fmk, caspase-8 inhibitor Z-IEHD-fmk, or normal saline for 96 h. Cell viability was then measured. The viability of untreated (without any cytokine and inhibitor) cells was set at 100%, and the relative viability of other samples was calculated accordingly. The experiments were performed in triplicate, and in at least two separated experiments. The bar indicates the standard error. **p<0.01, versus group treated with normal saline.

Article Snippet: A recombinant LIGHT soluble protein that only contained the extracellular region of human LIGHT and caspase inhibitors Z-DEVD-fmk, Z-IETD-fmk and Z-LEHD-fmk was purchased from R&D System (Minneapolis, MN, USA).

Techniques: Expressing, Nucleic Acid Electrophoresis, Western Blot, Control, Incubation, Saline

Journal: Oncotarget

Article Title: YM155 potently triggers cell death in breast cancer cells through an autophagy-NF-kB network

doi:

Figure Lengend Snippet: Ex vivo cultures of primary human breast tumors ( n = 19) were cultured 48 h with 50 nM YM155 or not treated (untreated). Samples were then analyzed for cancer cell morphology by Hematoxylin-Eosin-Saffron staining (upper panel) and active caspase-3 positivity by immunohistochemistry lower panel), as shown in A. for a YM155-responsive (left panel) or a resistant tumor (right panel). B. Data including 19 tumors are represented as % of carcinomatous cells positive in each specimen, in both untreated and YM155-treated conditions.

Article Snippet: YM155, Necrostatin-1 and BAY11-7085 were purchased from Selleck Chemicals (Houston, USA) and the pan-caspase inhibitor Q-VD-OPh from R&DSystems (Abingdon, UK).

Techniques: Ex Vivo, Cell Culture, Staining, Immunohistochemistry

Journal: Oncotarget

Article Title: YM155 potently triggers cell death in breast cancer cells through an autophagy-NF-kB network

doi:

Figure Lengend Snippet: A. MDA-MB321 or Cal51 cells were treated by YM115 at 40 nM and 70 nM respectively for 48 h and cleavage of caspase-3 and Bax proteins were evaluated by immunoblot analysis. The arrow on Bax immunoblot indicates a cleaved form of Bax with 18KDa MW. B. MDA-MB321, MCF-7 and Cal51 cells were first incubated with 10 μM of the pan-caspase inhibitor QVD-Oph (Q) and/or with 1 μM of the RIP1 inhibitor Necrostatin-1 (N) for 3 h then treated with respectively 50, 40 or 70 nM of YM155 for 48 h in presence of the above inhibitors (YM155) or not (untreated). Cell death assays were then performed and % of dead cells determined in each indicated condition. No significant inhibition of cell death was observed ( n = 3). C. A similar pretreatment with the autophagy inhibitors Chloroquine (25 μM) or 3-MA was applied to MDA-MB231, Cal51 or MCF-7 cells, followed by a 48 h-treatment by YM155 at above concentrations (YM155) or not (Un), before cell death evaluation. D. Similar culture conditions were applied before performing immunoblot analysis for LC3 evaluation. When indicated, Chloroquine pretreatment (25 μM) was realized 3 h before adding YM155. E. LC3 was evaluated by IHC in YM155-treated (+) at 40 nM for 48 h versus untreated (−) Cal51 cells.

Article Snippet: YM155, Necrostatin-1 and BAY11-7085 were purchased from Selleck Chemicals (Houston, USA) and the pan-caspase inhibitor Q-VD-OPh from R&DSystems (Abingdon, UK).

Techniques: Western Blot, Incubation, Inhibition

Journal: Structure (London, England : 1993)

Article Title: Epitope Topography of Agonist Antibodies to the Checkpoint Inhibitory Receptor BTLA

doi: 10.1016/j.str.2023.05.011

Figure Lengend Snippet: (A) Binding of BTLA agonist antibodies to a panel of BTLA mutants by ELISA as described in Methods. Lighter color indicates loss of binding signal to particular mutants. (B) Flow cytometric binding of BTLA agonist antibodies to BTLA mutants expressed in 293T cells. 293T cells transfected with various BTLA mutant-plasmids were used for the flow cytometry-based binding analysis. Cells were incubated with anti-BTLA clones (22B3, 6F4, and MIH26) for 40 min at 4°C. Appropriate secondary antibodies were used for detecting anti-BTLA agonist antibodies bound to mutant BTLA-expressing 293T cells. (C) Crystal structure of the human BTLA-HVEM complex: BTLA (brown) and HVEM (yellow) (2AW2.pdb). Residues that are important for HVEM and 22B3 binding are indicated. (D) Mimicry of HVEM binding by 22B3. Alignment of the 22B3:BTLA complex (green and cyan blue : brown) with the HVEM:BTLA complex (yellow : brown) shows that the CDR H3 (green sticks) is highly similar to a loop located on the CRD1 region of HVEM (yellow sticks).

Article Snippet: The HVEM and HVEM-BTLA reporter cell lines also have similar levels of NF-κB reporter activity as measured using phorbol ester as an agonist to directly activate the NF-κB reporter ( Fig. 5A right). (A) NF-κB reporter cell assay. (Left) Cell surface expression of HVEM in the HVEM 293T-NFκB cells (293T-NFκB-HVEM) and in the HVEM-BTLA co-expressing reporter cells (293T-NFκB-HVEM-BTLA) was determined by flow cytometry using an anti-HVEM monoclonal antibody (clone 94801, R&D Systems MAB356) detection of bound anti-HVEM. (Right) The NF-κB reporter system was compared between the HVEM-293 and HVEM-BTLA-293 cell lines.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Transfection, Mutagenesis, Flow Cytometry, Incubation, Clone Assay, Expressing

Journal: Structure (London, England : 1993)

Article Title: Epitope Topography of Agonist Antibodies to the Checkpoint Inhibitory Receptor BTLA

doi: 10.1016/j.str.2023.05.011

Figure Lengend Snippet: (A) Competitive binding of BTLA agonist mAbs 22B3, 25F7, and 23C8. BTLA-expressing 293T cells were used for flow cytometry-based analysis of competitive mAb binding. (Left) Competitive binding between 22B3 and 23C8. Cells were co-incubated with a fixed concentration of fluorescently labeled 22B3 (22B3-AF647) and graded concentrations of 23C8 mAb for 40 min at 4°C. (Middle) Competitive binding between 22B3 and 25F7. (Right) Competitive binding between 25F7 and 23C8. (B) Equilibrium binding analysis of 22B3, 25F7, and 23C8 to BTLA in 293T cells. BTLA-expressing 293T cells (293T-BTLA; magenta) and BTLA-HVEM co-expressing 293T cells (293T-BTLA-HVEM; green) were used for the flow cytometry-based binding analysis. Cells were incubated with graded concentrations of anti-BTLA mAb for 40 min at 4°C. and detected with an appropriate secondary antibody.

Article Snippet: The HVEM and HVEM-BTLA reporter cell lines also have similar levels of NF-κB reporter activity as measured using phorbol ester as an agonist to directly activate the NF-κB reporter ( Fig. 5A right). (A) NF-κB reporter cell assay. (Left) Cell surface expression of HVEM in the HVEM 293T-NFκB cells (293T-NFκB-HVEM) and in the HVEM-BTLA co-expressing reporter cells (293T-NFκB-HVEM-BTLA) was determined by flow cytometry using an anti-HVEM monoclonal antibody (clone 94801, R&D Systems MAB356) detection of bound anti-HVEM. (Right) The NF-κB reporter system was compared between the HVEM-293 and HVEM-BTLA-293 cell lines.

Techniques: Binding Assay, Expressing, Flow Cytometry, Incubation, Concentration Assay, Labeling

Journal: Structure (London, England : 1993)

Article Title: Epitope Topography of Agonist Antibodies to the Checkpoint Inhibitory Receptor BTLA

doi: 10.1016/j.str.2023.05.011

Figure Lengend Snippet: (A) NF-κB reporter cell assay. (Left) Cell surface expression of HVEM in the HVEM 293T-NFκB cells (293T-NFκB-HVEM) and in the HVEM-BTLA co-expressing reporter cells (293T-NFκB-HVEM-BTLA) was determined by flow cytometry using an anti-HVEM monoclonal antibody (clone 94801, R&D Systems MAB356) detection of bound anti-HVEM. (Right) The NF-κB reporter system was compared between the HVEM-293 and HVEM-BTLA-293 cell lines. The relative number of NF-κB reporter was determined in 293T-NFκB-HVEM and 293T-NFκB-HVEM-BTLA cells activated overnight with PMA (100 ng/ml) with the luciferase assay as described in the Materials and Methods section. NF-κB mediated luciferase activity was measured relative to the PMA activated positive control 293T-NFκB reporter cells. Each data point is the mean of 2 technical replicates. (B) Measurements of HVEM-mediated NF-κB activities were determined in the unstimulated reporter cells. Each data point is the mean of 2 technical replicates. (C) Antagonist activity of BTLA antibody 22B3 on HVEM signaling was determined in 293T-NFκB-HVEM-BTLA reporter cells. Each data point is the mean of 3 technical replicates.

Article Snippet: The HVEM and HVEM-BTLA reporter cell lines also have similar levels of NF-κB reporter activity as measured using phorbol ester as an agonist to directly activate the NF-κB reporter ( Fig. 5A right). (A) NF-κB reporter cell assay. (Left) Cell surface expression of HVEM in the HVEM 293T-NFκB cells (293T-NFκB-HVEM) and in the HVEM-BTLA co-expressing reporter cells (293T-NFκB-HVEM-BTLA) was determined by flow cytometry using an anti-HVEM monoclonal antibody (clone 94801, R&D Systems MAB356) detection of bound anti-HVEM. (Right) The NF-κB reporter system was compared between the HVEM-293 and HVEM-BTLA-293 cell lines.

Techniques: Expressing, Flow Cytometry, Luciferase, Activity Assay, Positive Control

Journal: Structure (London, England : 1993)

Article Title: Epitope Topography of Agonist Antibodies to the Checkpoint Inhibitory Receptor BTLA

doi: 10.1016/j.str.2023.05.011

Figure Lengend Snippet: (A) Proposed structural model of interaction between the HVEM-BTLA complex and anti-BTLA antibody 22B3. HVEM (salmon); BTLA 1 (magenta); BTLA 2 (yellow); 22B3 (green and blue). (B) Schematic illustration of the HVEM-BTLA cis -complex in the absence of 22B3. (C) Effect of 22B3 on HVEM-BTLA signaling. Diagrams show a top view of (B). Absence of 22B3 (left); bivalent binding of 22B3 (middle); monovalent binding of 22B3 (right).

Article Snippet: The HVEM and HVEM-BTLA reporter cell lines also have similar levels of NF-κB reporter activity as measured using phorbol ester as an agonist to directly activate the NF-κB reporter ( Fig. 5A right). (A) NF-κB reporter cell assay. (Left) Cell surface expression of HVEM in the HVEM 293T-NFκB cells (293T-NFκB-HVEM) and in the HVEM-BTLA co-expressing reporter cells (293T-NFκB-HVEM-BTLA) was determined by flow cytometry using an anti-HVEM monoclonal antibody (clone 94801, R&D Systems MAB356) detection of bound anti-HVEM. (Right) The NF-κB reporter system was compared between the HVEM-293 and HVEM-BTLA-293 cell lines.

Techniques: Binding Assay