Journal: Molecular medicine reports

Article Title: miR‑155 mediates inflammatory injury of hippocampal neuronal cells via the activation of microglia.

doi: 10.3892/mmr.2019.9917

Figure Lengend Snippet: Figure 2. miR‑155 increases the pro‑inflammatory cytokines expression in BV‑2 microglial cells. BV‑2 microglial cells were transfected with mimic control, miR‑155 mimics or treated with LPS. (A) ELISA and (B) reverse transcription‑quantitative polymerase chain reaction assays were performed to measure the expression levels of IL‑6, IL‑10, TNF‑α, TNF‑β and IDO1 in BV‑2 microglial cells. #P<0.05 vs. control; **P<0.01, ***P<0.001 vs. mimic control. miR, microRNA; LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor; IDO1, indoleamine 2,3‑dioxygenase 1.

Article Snippet: Commercially available ELISA kits were used to detect interleukin‐6 (IL‐6; cat. no. M6000B; R&D systems), IL‐10 (cat. no. M1000B; R&D systems), TNF‐α (cat. no. MTA00B; R&D systems), TNF‐β (cat. no. E‐EL‐M1210; Elabscience, Wuhan, China) and indoleamine 2,3-dioxygenase 1 (IDO1; cat. no. CSB‐EL010996MO; CUSABIO TECHNOLOGY LLC, Wuhan, China).

Techniques: Expressing, Transfection, Control, Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: miR‑155 mediates inflammatory injury of hippocampal neuronal cells via the activation of microglia.

doi: 10.3892/mmr.2019.9917

Figure Lengend Snippet: Figure 3. miR‑155 upregulates TNF‑α, TNF‑β and IDO1 expression in BV‑2 microglial cells. BV‑2 microglial cells were transfected with mimic control, miR‑155 mimics or treated with LPS. Western blotting was performed to evaluate the expression levels of TNF‑α, TNF‑β and IDO1 in BV‑2 microglial cells. #P<0.05 vs. control; **P<0.01, ***P<0.001 vs. mimic control. miR, microRNA; LPS, lipopolysaccharide; TNF, tumor necrosis factor; IDO1, indoleamine 2,3‑dioxygenase 1.

Article Snippet: Commercially available ELISA kits were used to detect interleukin‐6 (IL‐6; cat. no. M6000B; R&D systems), IL‐10 (cat. no. M1000B; R&D systems), TNF‐α (cat. no. MTA00B; R&D systems), TNF‐β (cat. no. E‐EL‐M1210; Elabscience, Wuhan, China) and indoleamine 2,3-dioxygenase 1 (IDO1; cat. no. CSB‐EL010996MO; CUSABIO TECHNOLOGY LLC, Wuhan, China).

Techniques: Expressing, Transfection, Control, Western Blot

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