Journal: The Journal of Experimental Medicine

Article Title: Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils

doi: 10.1084/jem.20180371

Figure Lengend Snippet: Destabilization of PCNA scaffold inhibits NADPH oxidase activity in vivo . (A–C) FACS analysis of cells in the peritoneal lavage 4 h after zymosan injection in mice treated with or without T2AA. Two independent sets of experiments were performed with a total of eight mice per group. (A) Total cell count. (B) Number of macrophages (F4/80 + ). (C) Number of neutrophils (Ly6G + ). (D) Effect of T2AA on ex vivo neutrophil NADPH oxidase activity measured by luminol-CL in peritoneal neutrophils stimulated or not with PMA. The data are CL peaks expressed as mean ± SEM ( n = 8). **, P < 0.01, ANOVA. (E and F) Inhibitory effect of T2AA on in vivo ROS production evaluated by L012-CL during zymosan-induced peritonitis. (E) Representative imaging of ROS production in individual mice. The pseudocolor heat maps represent photons/s/cm 2 /steradian. (F) Quantification of in vivo L012-CL in mice. The data are CL peaks expressed as mean ± SEM from five to six mice per group in two independent experiments; *, P < 0.05; ***, P < 0.001, ANOVA. (G and H) Ex vivo neutrophil NADPH oxidase activity measured by luminol-CL in neutrophils from WT or p21 −/− mice isolated from the peritoneal lavage 4 h after zymosan injection stimulated or not with PMA (as in D). (G) Representative kinetic analysis of NADPH oxidase activation in neutrophils from WT compared with p21 −/− mice. (H) The data are CL peaks expressed as mean ± SEM (from six mice per group in two independent experiments; *, P < 0.05, ANOVA).

Article Snippet: To measure in vivo neutrophil NADPH activation, the luminescent probe L012 (Sobioda) was prepared freshly right before the experiment by dissolving 2 mg L012/ml in sterile PBS and kept in the dark.

Techniques: Activity Assay, In Vivo, Injection, Cell Counting, Ex Vivo, Imaging, Isolation, Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils

doi: 10.1084/jem.20180371

Figure Lengend Snippet: Antiinflammatory effect of T2AA on TNBS-induced colitis. (A and B) Macroscopic changes following TNBS treatment are represented by colon weight/length ratio (A) and Wallace score (B) in the respective histograms. (C) Ameho score representative of histological lesions is shown in the histogram. The data (A–C) are mean ± SEM (**, P < 0.01; ***, P < 0.001, ANOVA) from six to eight mice per group in two independent experiments. (D) Photomicrographs are representative of H&E-stained slides (upper panels) and of MPO immunohistochemical labeling (lower panels) of paraffin-embedded colonic tissues recovered from controls, TNBS alone, and TNBS and T2AA. The scale bars correspond to 20 µm, magnification 200×. RG, regeneration; RE, reepithelization. (E) Colorimetric measurement of MDA in colon. The data are mean ± SEM ( n = 6); **, P < 0.01, ANOVA. (F and G) Inhibitory effect of T2AA on in vivo ROS production measured by L012-CL in TNBS-induced colitis mice. (F) Representative imaging of ROS production in individual mice with untreated TNBS-induced colitis (right panel) or T2AA-treated TNBS-induced colitis (left panel). This representative experiment shows the pseudocolor heat map representing photons/s/cm 2 /steradian. (G) Quantification of in vivo L012-CL from three to four mice per group using the percentages of L012-CL variation between 24 and 48 h. The data are expressed as mean ± SEM (*, P < 0.05, nonparametric Wilcoxon test).

Article Snippet: To measure in vivo neutrophil NADPH activation, the luminescent probe L012 (Sobioda) was prepared freshly right before the experiment by dissolving 2 mg L012/ml in sterile PBS and kept in the dark.

Techniques: Staining, Immunohistochemical staining, Labeling, In Vivo, Imaging