Journal: Science Advances

Article Title: Discovery of anti-inflammatory physiological peptides that promote tissue repair by reinforcing epithelial barrier formation

doi: 10.1126/sciadv.abj6895

Figure Lengend Snippet: ( A ) Schema of anti-JIPs Ab administration to Lgr5-EGFP-IRES-creERT2/tdTomato mice with DSS-induced colitis at the recovery stage. ( B ) Immunofluorescence of ZO-1 and DAPI in colon sections from mice treated as in (A). Scale bar, 20 μm. Similar results were obtained in two independent experiments. ( C ) DAI in accordance with assessment of stool consistency and fecal blood in ICR mice, in which 2% DSS was used for recovery ( n = 5 mice). ( D ) Quantification of the numbers of Gr-1–positive cells (9 to 12 images from five mice) treated as described in (C). ( E ) Relative intestinal permeability measured by plasma leakage of FITC-dextran (4 kDa) treated as described in (C) ( n = 5 mice from ctrl + preimmune or Ab, DSS + vehicle, n = 6 mice from ctrl + vehicle, recover + preimmune or Ab). ( F ) Silver staining of recombinant hA1AT incubated with or without MMP-1, MMP-8, or MMP-9. Similar results were obtained in two independent experiments. ( G ) Quantification of relative claudin-1 intensity at cell-cell boundaries of A431 cells treated with the indicated products or control HBSS ( n = 5 images from independent two samples). Similar results were obtained in two independent experiments. See fig. S7E. ( H ) Quantification of the relative claudin-1 intensity at cell-cell boundaries of A431 cells treated with CCM prepared from control, DSS-treated mice, or DSS-recovery mice in the presence or absence of GM6001, or control HBSS for 3 hours ( n = 5 images from independent two samples). Similar results were obtained in two independent experiments. See fig. S7F. Tukey’s test (C, D, G, and H) and the two-tailed t test (E). * P < 0.05, *** P < 0.001.

Article Snippet: Anti-JIP serum or preimmune serum was transfected into A431 cells by ProteoCarry (Funakoshi) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Permeability, Silver Staining, Recombinant, Incubation, Two Tailed Test

Journal: Science Advances

Article Title: Discovery of anti-inflammatory physiological peptides that promote tissue repair by reinforcing epithelial barrier formation

doi: 10.1126/sciadv.abj6895

Figure Lengend Snippet: ( A ) Immunofluorescence of rabbit IgG and ZO-1 in A431 cells transfected with anti-JIPs Ab or preimmune. Transfected cells were treated with JIP m35 (20 μM) or HBSS for 3 hours. Scale bar, 20 μm. ( B ) Liposome cosedimentation assay using biotinylated JIP m35 peptides or IgG (20 nmol each), which were bound to streptavidin-FITC ( n = 3 independent samples). ( C ) Relative barrier permeability measured by paracellular tracer flux analysis using FITC-dextran (4 kDa) in A431 cells transfected with G 12 and/or G 13 siRNA. See fig. S10B ( n = 3 independent samples). ( D ) TER measurements of EpH4 cells transfected with G 12 and/or G 13 siRNAs. See fig. S10D ( n = 3 independent samples). ( E ) Pull-down assay of A431 cells treated with biotinylated-JIP m35 , biotinylated-JIP m35-mut2 , or buffer using streptavidin-Sepharose. Precipitates were immunoblotted with anti-G 12 (upper), G 13 (bottom) antibodies. Asterisk indicates a nonspecific band. Quantitative value of each band was presented. ( F ) Measurement of G 13 activation by JIP m35 , JIP m35-mut1 , and JIP m35-mut2 ( n = 3 independent samples). JIP m35 was prepared at the indicating concentrations. ( G ) Measurement of G protein (G 13 , G i2 , G s , and G q ) activation by JIP m35 ( n = 3 independent samples). ( H ) Immunofluorescence of occludin, GFP, and G 13 in A431 cells transfected with GFP, G 13 , or G13Q226L. Scale bar, 10 μm. ( I ) Line scans represent the fluorescence intensity of occludin along the white arrow in (H), and a black arrowhead represents the position of cell junction. ( J ) The value represents the maximum value minus the minimum value of occludin intensity ( n = 9 cells). Data represent means ± SD (F and G); Tukey’s test (B and J) and Dunnett’s test (C, D, F, and G). n.s., not significant. * P < 0.05, ** P < 0.01, *** P < 0.001. Similar results were obtained in two independent experiments in (A) and (E).

Article Snippet: Anti-JIP serum or preimmune serum was transfected into A431 cells by ProteoCarry (Funakoshi) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Transfection, Permeability, Pull Down Assay, Activation Assay, Fluorescence

Journal: Journal of Bacteriology

Article Title: Identification and Characterization of a New Ferric Enterobactin Receptor, CfrB, in Campylobacter ▿ †

doi: 10.1128/JB.00478-10

Figure Lengend Snippet: Critical role of CfrB in Campylobacter-host interaction. (A) Effect of the CfrB mutation on colonization of chickens by C. jejuni JL11 and C. coli JL170. The experiment included two groups, and chickens in each group (15 birds) were inoculated with a 1:1 mixture of a specific Campylobacter strain and its isogenic cfrB mutant. Fecal samples were collected by cloacal swabs and used for CFU enumeration. Each bar represents the mean log transformed CFU of each strain in each group at the indicated days postinoculation (DPI). The broken line indicates the limit of detection. (B and C) Inactivation of CfrB abolishes C. coli JL170 colonization of chickens. The experiment included three groups of birds. The first two groups of birds were inoculated with JL170 or its isogenic cfrB mutant (JL170 CfrB−) (B), while the third group was infected with a 1:1 mixture of the two strains (C). For each group, six birds were euthanized and the cecal contents were collected at the indicated DPI. Each symbol indicates the log number of CFU/g of cecal contents for a single chicken. The horizontal bars indicate the means of groups at the indicated times. The broken line indicates the limit of detection. (D) Inhibitory effect of CfrB-specific antiserum on FeEnt-mediated growth promotion in Campylobacter. CfrB antiserum, MOMP antiserum, preimmune serum, or PBS was mixed with JL11 and JL170 cells for a modified growth promotion assay as described in Materials and Methods.

Article Snippet: Prior to the assay, the CfrB-specific rabbit antiserum, the preimmune control rabbit serum, and the major outer membrane protein (MOMP) rabbit antiserum specific for Campylobacter (kindly provided by Qijing Zhang, Iowa State University) were heat inactivated at 56°C for 30 min. To evaluate the inhibitory effect of anti-CfrB serum on CfrB function, 1 ml of MH broth grown log-phase culture of each Campylobacter strain was mixed with 1 ml of either CfrB antiserum, preimmune serum, MOMP antiserum, or phosphate-buffered saline (PBS) and then added to 23 ml of MH agar containing 20 μM DFO and poured into petri dishes for solidification.

Techniques: Mutagenesis, Transformation Assay, Infection, Modification