Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: Induction of apoptosis in 70Z/3 cells treated with 3CPA at 250 μ M and 500 μ M . ( A ) Cytochrome c release into the cytosol after exposure to 3CPA for indicated time periods measured by Western blot analysis. ( B ) Pro-caspase-9 processing determined in cytosolic fractions after exposure to 3CPA. Actin was used as an internal standard of the protein load and the results shown are representatives of two separate experiments. ( C ) Caspase inhibitors reduce 3CPA-induced apoptosis in 70Z/3 cells. Apoptosis, measured as AnnexinV + 7AAD − cells, was determined after exposure to 3CPA at 250 μ M (left) and 500 μ M (right) for 24 h. The pan-caspase inhibitor ZVAD-fmk (50 μ M , n =4), the caspase-9 inhibitor LEHD-fmk (50 μ M , n =3), or the caspase-8 inhibitor IETD-fmk (50 μ M , n =3) were added 1 h before treatment with 3CPA. The level of apoptosis is expressed relative to the level in cell cultures treated with the inhibitor only. Results show mean±s.d., and (*) indicates a statistical difference compared to 3CPA only (paired t -test, P <0.05).

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Western Blot

Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: Over-expression of Bcl-2 in 70Z/3 cells inhibits 3CPA-induced apoptosis. ( A ) FACS-analysis of 70Z/3 (upper panel) and 70Z/3 Bcl-2+ (lower panel) cells after exposure to 3CPA (500 μ M ) for 18 h. 7AAD negative (viable) cells were gated (gate A) and analysed for Annexin V + expression (% of gated cells). ( B ) Levels of Bcl-2 and cytochrome c expression were determined by Western blot analysis in mitochondrial enriched fractions after 18 h incubation with 3CPA at the indicated concentrations (30 μg protein per lane). ( C ) Pro-caspase-9 processing into active caspase-9 was determined in the cytosolic fraction after 18 h incubation with 3CPA at the indicated concentrations. Actin was used as an internal standard to control the amount of cytosolic protein loaded in each lane. Results shown are representatives of two independent experiments.

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Over Expression, Expressing, Western Blot, Incubation

Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: N-substituted benzamides induce cytochrome c release and activation of caspase-9 in HL60 cells. ( A ) Cytochrome c release induced by MCA (500 μ M ) and 3CPA (250 μ M ) in HL60 cells after 24 and 48 h, measured by Western blot analysis of mitochondria or cytosol fractions (30 μg protein/lane). Cells were incubated with etoposide for 6 and 18 h at 100 μ M as a positive control. ( B ) Activation of caspase-9 after treatment of HL60 cells with 3CPA at 250 and 500 μ M for 12 and 18 h (48 μg protein per lane). One representative experiment out of two is shown.

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Activation Assay, Western Blot, Incubation, Positive Control

Journal: Frontiers in Immunology

Article Title: Increased neutrophil extracellular trap formation in oligoarticular, polyarticular juvenile idiopathic arthritis and enthesitis-related arthritis: biomarkers for diagnosis and disease activity

doi: 10.3389/fimmu.2024.1436193

Figure Lengend Snippet: Demographic, clinical, and laboratory characteristics.

Article Snippet: To visualize whether TNF-α-induced NET formation was inhibited by TNF-α antagonists, neutrophils were seeded in the same way as described above and pretreated with a humanized anti-TNF-α antibody (adalimumab, Humira, Abbvie) (4 μg/mL, equivalent to the average serum concentration in the human body) for 15 minutes prior to TNF-α stimulation.

Techniques:

Journal: Frontiers in Immunology

Article Title: Increased neutrophil extracellular trap formation in oligoarticular, polyarticular juvenile idiopathic arthritis and enthesitis-related arthritis: biomarkers for diagnosis and disease activity

doi: 10.3389/fimmu.2024.1436193

Figure Lengend Snippet: Neutrophils derived from the peripheral blood of JIA patients increase NET formation in vitro , either spontaneously or in response to TNF-α or PMA. (A) Representative images of NETs released by neutrophils derived from at least 10 patients with JIA and 6 HCs spontaneously or in response to PMA or TNF-α. NETs were double immunostained for extracellular DNA (DAPI; blue) and MPO (MPO antibody; green), and NETs were identified by the presence of extracellular DNA stained with SYTOX-Green. Images were captured at 20× magnification; the scale bars represent 100 µm. (B) In a quantitative analysis of the above experiments, the percentages of NET-occupied areas in the total area in the JIA group were markedly higher than those in the HC group. (C–F) Quantitative assessment of NET formation in the o-JIA, p-JIA, and ERA groups by microplate assays. Neutrophils isolated from patients with o-JIA, p-JIA, and ERA and healthy controls were incubated for 6 hours under the conditions of (C) no intervention, (D) PMA (30 nM) stimulus, or (E) the addition of TNF-a (100 ng/ml). (F) The amounts of NETs were compared among the three subtypes of o-JIA, p-JIA, and ERA, respectively. The results are expressed as the fluorescence intensity of DNA in NETs. One-way ANOVA or the Kruskal−Wallis test was used to compare three or more groups. Student’s independent-sample t-test was used to compare the two groups. The bar graphs show the mean ± SEM or median with IQR. *p<0.05, **p<0.01, ***p<0.001, ns, nonsignificant; spontan, spontaneous.

Article Snippet: To visualize whether TNF-α-induced NET formation was inhibited by TNF-α antagonists, neutrophils were seeded in the same way as described above and pretreated with a humanized anti-TNF-α antibody (adalimumab, Humira, Abbvie) (4 μg/mL, equivalent to the average serum concentration in the human body) for 15 minutes prior to TNF-α stimulation.

Techniques: Derivative Assay, In Vitro, Staining, Isolation, Incubation, Fluorescence

Journal: Frontiers in Immunology

Article Title: Increased neutrophil extracellular trap formation in oligoarticular, polyarticular juvenile idiopathic arthritis and enthesitis-related arthritis: biomarkers for diagnosis and disease activity

doi: 10.3389/fimmu.2024.1436193

Figure Lengend Snippet: Associations of cf-DNA levels with immune-related parameters in the entire cohort of patients with JIA. (A) The concentrations of cf-DNA were strongly correlated with the number and percentage of neutrophils and CD19+ B cells, the expression of inflammatory markers (TNF-α, ESR, and hs-CRP), the levels of IgG, complement C3 and C4 in peripheral blood, but no correlation with IL-6 level. R values of Spearman or Pearson’s rank correlation and p values of their null hypothesis are shown. (B) ESR and TNF-α are independent variables by the multiple linear regression analysis.

Article Snippet: To visualize whether TNF-α-induced NET formation was inhibited by TNF-α antagonists, neutrophils were seeded in the same way as described above and pretreated with a humanized anti-TNF-α antibody (adalimumab, Humira, Abbvie) (4 μg/mL, equivalent to the average serum concentration in the human body) for 15 minutes prior to TNF-α stimulation.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Increased neutrophil extracellular trap formation in oligoarticular, polyarticular juvenile idiopathic arthritis and enthesitis-related arthritis: biomarkers for diagnosis and disease activity

doi: 10.3389/fimmu.2024.1436193

Figure Lengend Snippet: Effect of an anti-TNF-a antibody (adalimumab) on NET formation. Neutrophils isolated from JIA patients and HCs were pretreated with adalimumab (4 μg/ml) prior to the addition of TNF-α. (A) Representative images of TNF-α induced NETs and minimal NET formation after adalimumab intervention. The third column is an image of spontaneous NET formation as a negative control. (B) The percentage of the NET-occupied area under TNF-α stimulation was significantly decreased in the presence of adalimumab (n=18, 41.22% ± 2.351 vs. 20.39% ± 1.683, p < 0.0001). (C–F) The levels of NET release were measured via microplate assays to further evidence the ability of adalimumab to inhibit NET generation. (C) DPI can effectively inhibit PMA-induced NETosis. (D) Adalimumab (n=27) and DPI (n=16) effectively inhibited TNF-α-induced NET formation (n=27). (E) Comparison of the inhibitory effects of adalimumab and DPI on TNF-α-induced NET formation. (F) The inhibitory rate of adalimumab in NETosis by neutrophils from JIA (n=18) and HCs (n=9) under TNF-α stimulation. (G) The effect of TNF-α inhibitors on cf-DNA concentration and JADAS27 in JIA patients. The data were analyzed with paired-sample t test or unpaired-sample t test. The bar graphs show the mean ± SEM. *** p <0.001, **** p <0.0001, ns, nonsignificant.

Article Snippet: To visualize whether TNF-α-induced NET formation was inhibited by TNF-α antagonists, neutrophils were seeded in the same way as described above and pretreated with a humanized anti-TNF-α antibody (adalimumab, Humira, Abbvie) (4 μg/mL, equivalent to the average serum concentration in the human body) for 15 minutes prior to TNF-α stimulation.

Techniques: Isolation, Negative Control, Comparison, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Increased neutrophil extracellular trap formation in oligoarticular, polyarticular juvenile idiopathic arthritis and enthesitis-related arthritis: biomarkers for diagnosis and disease activity

doi: 10.3389/fimmu.2024.1436193

Figure Lengend Snippet: ROS levels were measured ex vivo using a FACSCalibur flow cytometer. (A) The exposure of neutrophils to TNF-α increased in the production of ROS. (B) Baseline ROS levels in neutrophils derived from JIA patients were higher than those in HC-derived neutrophils (n = 20; p = 0.0074). The paired-sample t test was used to analyze all the data. * p < 0.05, ** p <0.01.

Article Snippet: To visualize whether TNF-α-induced NET formation was inhibited by TNF-α antagonists, neutrophils were seeded in the same way as described above and pretreated with a humanized anti-TNF-α antibody (adalimumab, Humira, Abbvie) (4 μg/mL, equivalent to the average serum concentration in the human body) for 15 minutes prior to TNF-α stimulation.

Techniques: Ex Vivo, Flow Cytometry, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Increased neutrophil extracellular trap formation in oligoarticular, polyarticular juvenile idiopathic arthritis and enthesitis-related arthritis: biomarkers for diagnosis and disease activity

doi: 10.3389/fimmu.2024.1436193

Figure Lengend Snippet: The expression levels of NET-associated signaling components. (A) The phosphorylation of PI3K and AKT in neutrophils purified from JIA patients and HCs before and after TNF-α stimulation (p-PI3K n = 10, p = 0.0008; p-AKT, n = 6, p = 0.0313). The paired-sample t test and Wilcoxon test were used to analyze the data. (B) Comparison of phosphorylated PI3K and AKT protein levels in neutrophils derived from JIA patients and HCs after TNF-α stimulation (n = 5, p = 0.7340; n = 3, p = 0.3386, respectively). (C) Phosphorylation of PI3K (15 HCs vs. 18 JIA patients, p = 0.0222) and AKT (n = 10 p = 0.0135) and the protein expression of MPO (n = 15, p = 0.0012) in neutrophils derived from JIA patients and HCs. (D) MAPK-CDK and ERK1/2 phosphorylation was assessed (6 HCs vs. 7 JIA patients p = 0.0239). (A, C, D) Representative western blots. The Mann−Whitney U test was used to compare two groups. Stim, stimulated; unstim, unstimulated. The bar graphs show the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, ns, nonsignificant.

Article Snippet: To visualize whether TNF-α-induced NET formation was inhibited by TNF-α antagonists, neutrophils were seeded in the same way as described above and pretreated with a humanized anti-TNF-α antibody (adalimumab, Humira, Abbvie) (4 μg/mL, equivalent to the average serum concentration in the human body) for 15 minutes prior to TNF-α stimulation.

Techniques: Expressing, Phospho-proteomics, Purification, Comparison, Derivative Assay, Western Blot, MANN-WHITNEY