Fig 1.
Qualitative and quantitative analysis of NETs formation induced by treatment of neutrophil-like cells with A23187.
(A and B) Representative images obtained by fluorescence microscopy of neutrophil-like cells stained with Sytox Green cell-impermeable nucleic acid dye in basal conditions (A) and after stimulation with 25 μM A23187 for 4 h. Scale bar, 20 μm (B). Representative image obtained by fluorescence microscopy at higher magnification showing morphological details of NETs stained with antibody recognizing Cit H3. Scale bar, 5 μm (C). Results of three independent fluorescence plate reader assays in unstimulated and stimulated neutrophil-like cells expressed as percentage of total DNA released from lysed cells (D).
Fig 2.
Levels of total and citrullinated histone H3 in NETs.
Western blot analysis of total and citrullinated histone H3 (Cit H3) in conditioned media (A) and in NETs-enriched suspension (B) from stimulated neutrophil-like cells compared to the corresponding negative control.
Fig 3.
Integrin expression and cell adhesion to NETs.
(A and B) Relative levels of α5β1 and ανβ3 integrins in human K562 cells (A) and its derived clone K562ανβ3 (B) were determined by flow cytometry analysis using primary monoclonal antibodies HA5 (blue histograms) and LM609 (green histograms) recognizing α5β1 and ανβ3 integrins, respectively, and compared to the corresponding negative control (red histograms). Two independent experiments were performed for each cell line and antibody. (C and D) Cell adhesion assays to NETs in K562 (C) and K562ανβ3 (D) cells. Three independent experiments were performed using different NETs stocks obtained from the stimulation of newly differentiated HL-60 cells in the presence of 10% FBS. Results are expressed as percentage of adherent cells compared to the total number of added cells and mean ± SD of three independent experiments is reported for each condition. Statistical significant differences between NETs coated and each experimental condition is indicated by symbols *, ** and ***, meaning p<0.05, p< 0.01 and p<0.001, respectively.
Table 1.
Adhesion of K562 and K562ανβ3 cells to NETs.
Fig 4.
Levels of fibronectin and interaction with histone H3 in NETs.
(A) Levels of fibronectin were determined by Western blot analysis performed on samples of conditioned media of neutrophil-like cells stimulated or not with increasing concentration of A23187 in the presence (left, 50 μg of protein per lane) or absence (right, 5 μg of protein per lane) of serum. (B) Western blot analysis of samples of NETs preparations obtained by stimulating neutrophil-like cells with 25 μM A23187 for 4 h in the presence (left, 50 μg of protein per lane) or absence (right, 5 μg of protein per lane) of serum compared to the corresponding negative control from unstimulated cells. Gels were stained with Imperial protein stain to obtain SDS-PAGE patterns of protein content and ensure equal loading. (C) Immunoprecipitation of conditioned media of unstimulated and A23187-stimulated neutrophil-like cells was performed using anti-fibronectin antibody followed by SDS-PAGE of the immunoprecipitated proteins and immunoblotting with the indicated antibodies.
Fig 5.
Co-localization of citrullinated histone H3 with myeloperoxidase and fibronectin in NETs.
(A) Representative images obtained with confocal microscopy showing co-localization of MPO (green) and Cit H3 (red) in neutrophil-like cells stimulated in serum-free conditions with 25 μM A23187 for 4 h (upper panels) or in cell-free NETs enriched suspension (lower panels). Merged images showed the co-localization of MPO and Cit H3 confirming the typical features of NETs. (B) Representative images obtained with confocal microscopy showing co-localization of fibronectin (green) and Cit H3 (red) in neutrophil-like cells stimulated in serum-free conditions with 25 μM A23187 for 4 h (upper and middle panels) or in cell-free NETs enriched suspension (lower panels). Merged images showed a clear co-localization of Cit H3 and fibronectin in the structure of NETs at early (upper panels) and late (middle panels) phases of the process coexisting in different fields as well as in NETs after their isolation procedures (lower panels).