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Neutrophils and cancer-associated thrombosis
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Figure 2. Analysis of thrombus constituents. (A) Representative western blot of Ly6G, β-actin, histone H3, and citrullinated histone H3 (H3Cit) in thrombi from control and tumor-bearing mice. Arrows on the left of the panel indicate the target proteins and the size of the molecular weight markers are shown on the right. (B-E) Levels of the different markers were quantified by densitometric analysis. (F) The H3Cit/H3 ratio for thrombi from control and tumor-bearing mice are shown. AU: arbitrary unit. Eight mice were used for each group. Data were analyzed with the unpaired t-test or the Mann-Whitney U-test depending on the type of data distribution. *P<0.05, **P<0.01, ***P<0.001.
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Figure 3. Analysis of thrombi by immunofluorescence. (A, B) Thrombus sections from control (A) and tumor-bearing (B) mice were stained for cell-free DNA (TOTO-3, blue), citrullinated histone H3 (H3Cit; immunostained, green) and fibrin (immunostained, red) and examined with confocal laser scanning microscopy (Zeiss LSM 710, Carl Zeiss, Jena, Germany). (C, D) The area occupied by the DNA (C) and H3Cit (D) signal was quantified in 15 randomly selected images from each thrombus shown with the same color. Lines indicate the median values of the bottom, median and top quartiles calculated from the data of 90 images from six animals of the control group (shown in dif- ferent colors) and 150 images from ten animals of the tumor group (shown in different colors). The statistical analysis was performed using 90 or 150 input data according to the two-step procedure described in the Online Supplement (Bootstrap Kuiper test P<0.001 for all three quartiles of the datasets in panels C and D).
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