Fig 1: GSK484-attenuated PAO-mediated ALI in mice.WT mice were injected i.p. with GSK484 before the exposure to PAO on skin and sacrificed after 6 hours. (A) H&E staining, (B) lung injury score (n = 5/group), and (C) dsDNA in BAL fluid control, GSK484, PAO, and PAO and GSK484 groups (n = 5/group). Scale bar: 100 µm. (D) IHC staining of lung sections for Cit-H3 from control, GSK484, PAO, and PAO and GSK484 groups. Scale bar: 100 µm. Lower panel original magnification, ×2500. (E) Measurements of DAB intensity from IHC for Cit-H3. An average measurement from 5 mice was calculated from the average of 8 images. (F) Soluble vWF levels in serum (n = 5/group). (G) Western blot analysis for PAD4 and Cit-H3 expression in lung homogenates (n = 3). (H) Western blot densitometry for Cit-H3 and PAD4. The Cit-H3 and PAD4 densitometry was normalized with ß-actin. (I) vWF levels in the serum (n = 5/group). (J) Fold changes in the protein leak in BAL fluid from control, PAO, PAD4–/–, PAD4–/– PAO, GSK484, and GSK484 and PAO groups (n = 5–6 mice/group). Statistics: (B and C) box-and-whiskers plots display median, and whiskers show maximum to minimum value. Each symbol represents individual mouse. **P < 0.01. Data were analyzed by using 1-way ANOVA followed by Tukey’s multiple comparisons test. In graphs (E and G–J), data are shown as the mean ± SEM. *P < 0.05,**P < 0.01.
Fig 2: Single exposure of PAO on the skin results in ALI.PAO (150 µg/mouse in 2 cm2 area) was applied to the skin of shaved C57BL/6J mice. These mice were sacrificed after 6 hours. (A) Micro-CT scan view at thoracic vertebrae shown in control and PAO group. (B) Micro-CT scan analysis for density of the lung (n = 7/group). (C) H&E staining images of lung sections (n = 5–6/group). Scale bar: 100 µm. (D) ALI score (control, n = 5 and PAO, n = 6). (E) Wet-to-dry weight ratio of mouse lungs (n = 5/group). (F) Protein levels in BAL fluid (n = 5/group). (G) Neutrophil elastase activity in BAL fluid (n = 5/group). (H) mRNA levels for inflammatory genes in BAL cell pellets (n = 4/group). (I) Cytokine protein array for acute inflammation in whole lung lysate of control and PAO groups. The selected cytokine levels are represented in the graph after normalization with the control group (n = 3 for each group). (J) Immunohistochemistry (IHC) for vWF in lung sections (n = 5/group). Scale bar: 100 µm. (K) Soluble vWF levels in serum (n = 5/group). Error bars are shown as the mean ± SEM. *P < 0.05, **P < 0.01. Statistics: 2-tailed t test. All experiments were repeated 3 times.
Fig 3: Lewisite skin burn instigates ALI.Lewisite (5.0 mg/kg) was applied to the skin of hairless Ptch1+/- SKH-1 mice. Mice were sacrificed after 6 hours. (A) H&E images of lung sections (n = 5/group). (B) ALI scores from a blinded pathologist based on a standardized scoring system from the American Thoracic Society. Scores are continuous between 0 and 1, with 0 representing no injury and 1 representing severe ALI (n = 5/group). (C) BAL cell pellets (original magnification, ×400) stained with hematoxylin (nuclear stain). (D) Percentage of neutrophils counted in BAL cell pellets (n = 5/group). (E) Protein concentrations in BAL fluid (n = 5/group). (F) Immunohistochemistry staining of lung sections for vWF (n = 5/group). Scale bar: 100 µm. (G) Soluble vWF levels in serum (n = 5/group). All experiments were repeated 3 times. (B, D, E, and G) Each dot represents an individual mouse; error bars indicate the mean ± SEM. *P < 0.01. Statistics: 2-tailed t test.
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