Journal: Cell Death & Disease

Article Title: Membrane lymphotoxin-α 2 β is a novel tumor necrosis factor (TNF) receptor 2 (TNFR2) agonist

doi: 10.1038/s41419-021-03633-8

Figure Lengend Snippet: A Scheme of possible trimers formed upon coexpression of LTα and a GpL fusion protein of soluble LTβ (GpL-sLTβ). Please note, LTβ trimers are expressed but do not bind to LTβR. B HEK293T cells were transiently transfected with empty vector (EV) or an expression plasmid encoding LTβRed-GPI. Cells were then incubated for 1 h with 1 µg/ml of cell culture supernatant, transfected with GpL-sLTβ or a mixture of LTα and GpL-sLTβ-encoding expressions plasmids. Cell bound molecules were quantified by measuring GpL activity. Shown are results from three independent experiments. C HEK293T cells were transiently transfected with empty vector (EV) or expression plasmids encoding TNFR1-GPI, TNFR2-GFP, or LTβRed-GPI and were preincubated the next day as indicated for 30 min with 10 µg/ml TNF, 10 µg/ml of a TNFR2-specific antibody, TNFR1- or LTβR-specific Fabs. Cells were then incubated for 1 h with a cell culture supernatant of cells transfected with a 1:1 mixture of LTα and GpL-sLTβ-encoding expressions plasmid (final ligand concentration 20 ng/ml). Finally, cell bound molecules were quantified by measuring GpL activity. Shown are results from 10 (EV), 3 (TNFR1-GPI), 6 (TNFR2-GPI) and 8 (LTβR-GPI) independent experiments. D HeLa-TNFR2 and Kym-1 cells were preincubated for 30 min with 10 µg/ml anti-TNFR1-Fab, anti-TNFR2, anti-LTβR-Fab, or a combination of all three molecules and were then evaluated with respect to binding of GpL-sLTβ-containing ligand species as in (C). Shown are the results from six different experiments. *** p < 0.001; ** p < 0.01; * p < 0.05; repeated-measures ANOVA.

Article Snippet: Antibodies used in this study were purchased from the following suppliers: BD Biosciences, NJ, USA (anti-PARP, 551025; anti-RIPK1, 610459), Cell Signaling, MA, USA (anti-p-RIPK1, 65746 S; anti-Caspase-9, 9502 S), Enzo Life Sciences, Germany (anti-Caspase-8, ADI-AAM-118-E), Santa Cruz Biotechnology, Santa Cruz, CA, USA (anti-TNFβ (TNFβ = LTα) (E-6), sc-28345), Sigma-Aldrich, Germany (anti-mouse IgG (whole molecule), R-PE-labeled, P9670; anti-β-Aktin, A1978) and Thermo Fisher Scientific, MA, USA (anti-TNFα, PE-labeled, #12-7349-82).

Techniques: Transfection, Plasmid Preparation, Expressing, Incubation, Cell Culture, Activity Assay, Concentration Assay, Binding Assay

Journal: Cell Death & Disease

Article Title: Membrane lymphotoxin-α 2 β is a novel tumor necrosis factor (TNF) receptor 2 (TNFR2) agonist

doi: 10.1038/s41419-021-03633-8

Figure Lengend Snippet: A Scheme of the TNFRed–GpL fusion proteins used in (B). B HEK293T cells were transfected with empty vector (EV) or a mixture of LTα- and memLTβ-encoding expression plasmids. Next day, binding of 500 ng/ml TNFR1ed–GpL, TNFR2ed–GpL, and LTβRed–GpL was analyzed in the presence and absence of 20 µg/ml TNF. Data shown are technical replicates of one representative experiment of three independent experiments.

Article Snippet: Antibodies used in this study were purchased from the following suppliers: BD Biosciences, NJ, USA (anti-PARP, 551025; anti-RIPK1, 610459), Cell Signaling, MA, USA (anti-p-RIPK1, 65746 S; anti-Caspase-9, 9502 S), Enzo Life Sciences, Germany (anti-Caspase-8, ADI-AAM-118-E), Santa Cruz Biotechnology, Santa Cruz, CA, USA (anti-TNFβ (TNFβ = LTα) (E-6), sc-28345), Sigma-Aldrich, Germany (anti-mouse IgG (whole molecule), R-PE-labeled, P9670; anti-β-Aktin, A1978) and Thermo Fisher Scientific, MA, USA (anti-TNFα, PE-labeled, #12-7349-82).

Techniques: Transfection, Plasmid Preparation, Expressing, Binding Assay

Journal: Cell Death & Disease

Article Title: Membrane lymphotoxin-α 2 β is a novel tumor necrosis factor (TNF) receptor 2 (TNFR2) agonist

doi: 10.1038/s41419-021-03633-8

Figure Lengend Snippet: A Scheme of single-chain encoded memLTαβ variants and memTNF. B Flow cytometry analysis of Flp-In HEK293 transfectants stably expressing mem(sc)LTα 2 β and mem(sc)LTαβ 2 and of a CHO transfectant stably expressing a non-cleavable mutant of memTNF. Empty vector (EV) transfected cells served as negative controls. C The indicated Flp-In HEK293 and CHO-Δ(1-12)TNF cells were analyzed with respect to binding of TNFR1ed-GpL, TNFR2ed-GpL, and LTβRed-GpL. One representative experiment of three independent experiments are shown. D HeLa, HeLa-TNFR2, and HeLa-TNFR2-TNFR1 KO cells were cocultured 1:1 (50,000 cells each) with the indicated ligand transfectants. Cell culture supernatants were analyzed the next day for their IL8 content by ELISA. Shown are results from three different experiments. *** p < 0.001; ** p < 0.01; n.s., not significant, repeated-measures ANOVA.

Article Snippet: Antibodies used in this study were purchased from the following suppliers: BD Biosciences, NJ, USA (anti-PARP, 551025; anti-RIPK1, 610459), Cell Signaling, MA, USA (anti-p-RIPK1, 65746 S; anti-Caspase-9, 9502 S), Enzo Life Sciences, Germany (anti-Caspase-8, ADI-AAM-118-E), Santa Cruz Biotechnology, Santa Cruz, CA, USA (anti-TNFβ (TNFβ = LTα) (E-6), sc-28345), Sigma-Aldrich, Germany (anti-mouse IgG (whole molecule), R-PE-labeled, P9670; anti-β-Aktin, A1978) and Thermo Fisher Scientific, MA, USA (anti-TNFα, PE-labeled, #12-7349-82).

Techniques: Flow Cytometry, Stable Transfection, Expressing, Transfection, Mutagenesis, Plasmid Preparation, Binding Assay, Cell Culture, Enzyme-linked Immunosorbent Assay