Journal:

Article Title: High-Level Replication of Human Immunodeficiency Virus in Thymocytes Requires NF-?B Activation through Interaction with Thymic Epithelial Cells

doi:

Figure Lengend Snippet: TNF and, to a lesser extent, IL-1 are involved in the permanently induced NF-κB activity present in thymocytes during the coculture with TEC. Thymocytes were freshly isolated (lanes 1 and 2) or cultured for 45 h (lanes 3 to 11) either alone (lane 3 [Control thymocytes]) or in the presence of TEC under conditions avoiding contact (lanes 4 to 9) or allowing contact (lanes 10 and 11). During the coculture, the cells were either left untreated (lane 4 [Control coculture, 45 hours] and lane 10 [Control coculture]) or were treated at the start of the coculture with antibodies respectively raised against TNF (lane 5), IL-6 (lane 6), and IL-1β (lane 8), with the antagonist of the IL-1 receptor (lane 7), or with αTNF plus IL-1 ra plus αIL-6 (lanes 9 and 11). Whole-cell extracts from these various samples were incubated with a 32P-labeled oligonucleotide representing the HIV LTR-derived κB motif. Competition (comp [lane 1]) with a 40-fold molar excess of unlabeled oligonucleotide was used to confirm the specificity of the DNA-binding activity detected. NF-κB activity is indicated. The positions of two nonspecific (n.s 1 and n.s 2) binding activities are indicated. The positions of two nonspecific (n.s 1 and n.s 2) binding activities are indicated. This experiment is representative of three independent experiments, each carried out on a different thymus.

Article Snippet: MAb against IL-1β (B-A15) was purchased from Diaclone Research (Besançon, France).

Techniques: Activity Assay, Isolation, Cell Culture, Incubation, Labeling, Derivative Assay, Binding Assay

Journal:

Article Title: High-Level Replication of Human Immunodeficiency Virus in Thymocytes Requires NF-?B Activation through Interaction with Thymic Epithelial Cells

doi:

Figure Lengend Snippet: TNF and, to a lesser extent, IL-1 induce NF-κB activity in thymocytes, and IL-7 is required for this effect. Whole-cell extracts were prepared from freshly isolated thymocytes (panel A, lane 1) or from thymocytes cultivated for 30, 60, or 45 h. (A) During the indicated culture times, thymocytes were either left untreated (lane 1, control for freshly isolated; lane 2, control 30 h; and lane 5, control 60 h) or were stimulated with either TNF (lanes 3 and 6), IL-1β (lanes 4 and 7), or TNF in the presence of TEC CM (TEC CM + TNF) (lane 9). TEC conditioned medium was also added alone (lane 8 [TEC CM]). The data shown here are representative of three independent experiments carried out on three thymuses. (B) Thymocytes were cultured for 45 h either untreated (lane 1, control 45 h), with TNF (lane 2), with TEC CM (lane 3), or with TNF in the presence of TEC CM (TEC CM + TNF) preincubated (lane 5) or not (lane 4) with antibody against IL-7. In lanes 6 to 8, the thymocytes were left untreated for 6 h (lane 6) or incubated with an antibody against IL-7Rα (lane 7) or with an IgG1 control serum (lane 8) before being cocultured with TEC for 45 h. The data shown here are representative of two independent experiments carried out on two thymuses. (C) Cells were left untreated for the 45 h of the culture (lane 1, control 45 h) or treated with IL-1β (lane 2), TNF (lane 3), IL-1β plus TNF plus IL-6 plus GM-CSF (lane 4), IL-7 (lane 5), IL-7 plus IL-1β (lane 6), or IL-7 plus TNF (lane 7). This experiment is representative of three independent experiments carried out on three thymuses. (D) Thymocytes were left untreated (lane 1, control) or stimulated with IL-7 (lane 2) or IL-7 in presence of anti-TNF and Il-1ra (lane 3). Whole-cell extracts were incubated with a 32P-labeled oligonucleotide representing κB motif. NF-κB activity is indicated. This experiment is representative of two independent experiments, each carried out on a different thymus.

Article Snippet: MAb against IL-1β (B-A15) was purchased from Diaclone Research (Besançon, France).

Techniques: Activity Assay, Isolation, Cell Culture, Incubation, Labeling

Journal: Neural Regeneration Research

Article Title: Lithium promotes recovery after spinal cord injury

doi: 10.4103/1673-5374.327348

Figure Lengend Snippet: Lithium suppresses activation of inflammatory cytokines in the spinal cord of rats with SCI . Sham group: No spinal cord injury or treatment; SCI group: spinal cord injury only; LiCl group: lithium treatment after spinal cord injury. (A–C) TNF-α (A), IL-1β (B), and IL-6 (C) expression in the spinal cord were detected by enzyme-linked immunosorbent assay. (D) Representative immunohistochemical staining for IL-1β 7 days after surgery. The number of IL-1β-positive cells was higher in the LiCl group than in the sham group, but lower than in the SCI group. Black arrows indicate IL-1β-positive cells. Scale bars: 50 μm. (E) Quantification of IL-1β-positive cells 7 days after surgery. (F, G) TNF-α, IL-1β, and IL-6 expression. Data are shown as mean ± SD ( n = 6). * P < 0.05, vs . sham group; # P < 0.05, vs . SCI group (one-way analysis of variance followed by the least significant difference test). IL-1β: Interleukin-1β; IL-6: interleukin-6; LiCl: lithium chloride; SCI: spinal cord injury; TNF-α: tumor necrosis factor-alpha.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with rat monoclonal primary antibodies against IL-6 (1:1000, Acris Antibodies GmbH, Herford, Germany; Cat# SM1695PX, RRID:AB_1004115), IL-1β (1:100, Miltenyi Biotec, Bergisch Gladbach, Germany; Cat# 130-125-220, RRID:AB_2889697), TNF-α (1:100, Miltenyi Biotec; Cat# 130-102-386, RRID:AB_2661141), NLRP3 (1:1000, MyBioSource, San Diego, CA, USA; Cat# MBS604215, RRID:AB_10911109), ASC (1:2000, Gabriel Núñez, University of Michigan, Ann Arbor, MI, USA; Cat# GN-ASC, RRID:AB_2750645), pro-Caspase-1 (1:1000, ProSci, San Diego, CA, USA; Cat# 48-495, RRID:AB_1945485), Caspase-1 (1:1000, Acris Antibodies GmbH; Cat# AP06610PU-N, RRID:AB_1611115), GSDMD (1:100, Thermo Fisher Scientific; Cat# A305-736A-M, RRID:AB_2782894), and IL-18 (1:100, DSHB, Iowa City, IA, USA; Cat# CPTC-IL18-2, RRID:AB_1553716), followed by incubation with horseradish peroxidase-conjugated rabbit anti-rat IgG (1:1000, Biorbyt, Cat# orb21564, RRID:AB_10932535) at 20°C for 1 hour, and finally stained with a diaminobenzidine solution.

Techniques: Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining

Journal: Neural Regeneration Research

Article Title: Lithium promotes recovery after spinal cord injury

doi: 10.4103/1673-5374.327348

Figure Lengend Snippet: Lithium attenuates inflammatory, oxidative, and pyroptotic injury in PC12 cells subjected to OGD . Con group: untreated PC12 cells; OGD group: PC12 cells subjected to OGD; OGD + LiCl group: PC12 cells subjected to OGD and treated with LiCl; OGD + LiCl + ATRA group: PC12 cells subjected to OGD and treated with LiCl and the Nrf2 inhibitor ATRA. (A–I) TNF-α (A), IL-1β (B), IL-6 (C), CAT (D), GSH-Px (E), LDH (F), SOD (G), MDA (H), and T-AOC (I) expression as detected by ELISA. (J–N) Flow cytometry analysis of the percentage of pyroptotic PC12 cells. Data are shown as mean ± SD ( n = 6). * P < 0.05, vs . control group; # P < 0.05, vs . OGD group; & P < 0.05, vs . OGD + LiCl group (one-way analysis of variance followed by least significant difference test). ATRA: All-trans retinoic acid; CAT: catalase; Con: control; GSH-Px: glutathione peroxidase; IL-1β: interleukin-1β; IL-6: interleukin-6; LiCl: lithium chloride; LPO: lipid peroxide; MDA: malondialdehyde; OGD: oxygen glucose deprivation; SOD: superoxide dismutase; T-AOC: total antioxidant capacity; TNF-α: tumor necrosis factor-alpha.

Article Snippet: Subsequently, the membranes were incubated overnight at 4°C with rat monoclonal primary antibodies against IL-6 (1:1000, Acris Antibodies GmbH, Herford, Germany; Cat# SM1695PX, RRID:AB_1004115), IL-1β (1:100, Miltenyi Biotec, Bergisch Gladbach, Germany; Cat# 130-125-220, RRID:AB_2889697), TNF-α (1:100, Miltenyi Biotec; Cat# 130-102-386, RRID:AB_2661141), NLRP3 (1:1000, MyBioSource, San Diego, CA, USA; Cat# MBS604215, RRID:AB_10911109), ASC (1:2000, Gabriel Núñez, University of Michigan, Ann Arbor, MI, USA; Cat# GN-ASC, RRID:AB_2750645), pro-Caspase-1 (1:1000, ProSci, San Diego, CA, USA; Cat# 48-495, RRID:AB_1945485), Caspase-1 (1:1000, Acris Antibodies GmbH; Cat# AP06610PU-N, RRID:AB_1611115), GSDMD (1:100, Thermo Fisher Scientific; Cat# A305-736A-M, RRID:AB_2782894), and IL-18 (1:100, DSHB, Iowa City, IA, USA; Cat# CPTC-IL18-2, RRID:AB_1553716), followed by incubation with horseradish peroxidase-conjugated rabbit anti-rat IgG (1:1000, Biorbyt, Cat# orb21564, RRID:AB_10932535) at 20°C for 1 hour, and finally stained with a diaminobenzidine solution.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Control