Fig 2: Immunofluorescent staining of HMGB1. Immunofluorescent co-staining identifies colocalization of (a) HMGB1, CD66b and DNA (by DAPI) and spatial proximity of (b) HMGB1 and CD42b. Representative magnifications illustrate the spatial coexistence of HMGB1 and CD66b, whereas CD42b was detected within the interspace of HMGB1-positive areas and has only marginal contact with HMGB1. Scale bars: (a,b) 100 µm, (c,d) 50 µm. HMGB 1 = high mobility group box 1, CD = cluster of differentiation, DAPI = 4,6-diamidino-2-phenylindole.

Fig 3: Immunohistochemical analyses of human liver biopsies suggest intrahepatic formation of NETs. Representative image of a human postreperfusion liver needle biopsy stained for (A) CD66b and (B and C) NE. Scale bars indicate (A and B) 200 µm and (C) 50 µm.

Fig 4: Antibiotic treatment reduces the number of osteoclasts and immune cells at the apex area of pulp exposed tooth. a Representative images and quantification of TRAP + osteoclast number at the apex of pulp exposed tooth. b, c Representative images and quantification of immunofluorescent staining for CD66b+ (b) and CD3+ (c) cells at the apex area of pulp exposed tooth. Dashed lines outline the area where CD66b+ and CD3+ cells were counted. All quantified data represent mean ± SEM (n = 8). *P < 0.05; ***P < 0.001. Scale bar: 100 μm

Fig 5: NETs levels are elevated in HCC, especially in HBV‐positive HCC. (A‐B) The representative images of IF staining for CD66b (A) in tissue sections from HCC patients, HBV‐HCC patients and their matched para‐carcinoma tissues. The IOD of CD66b (B) IF‐stained materials were analyzed utilizing the Image Pro Plus software. Data from histomorphometric analysis are presented as mean ± standard deviation calculated from 8 HCC patients, 8 HBV‐HCC patients and their matched para‐carcinoma tissues. (C‐D) The representative images of IF staining for CitH3 (C) in tissue sections from HCC patients, HBV‐HCC patients and their matched para‐carcinoma tissues. The IOD of CitH3 (D) IF‐stained materials were analyzed utilizing the Image Pro Plus software. Data from histomorphometric analysis are presented as mean ± standard deviation calculated from 8 HCC patients, 8 HBV‐HCC patients and their matched para‐carcinoma tissues. (E) Western blot analysis for CitH3 expression in tissue sections from 8 HCC patients, 8 HBV‐HCC patients and their matched para‐carcinoma tissues. The fold change of densitometric ratios was normalized to the β‐actin and then compared to the control and was shown on the below panel. (F) Western blot analysis for CitH3 expression in peripheral neutrophils from 6 healthy controls, 6 HCC patients and 6 HBV‐HCC patients. The fold change of densitometric ratios was normalized to the β‐actin and then compared to the control and was shown on the right panel. (G) The representative images of IF staining for CitH3 in human neutrophils from healthy controls, HCC patients and HBV‐HCC patients. (H) The IOD of CitH3 IF‐stained materials were analyzed utilizing the Image Pro Plus software. Data from histomorphometric analysis are presented as mean ± standard deviation calculated from 6 healthy controls, 6 HCC patients and 6 HBV‐HCC patients. (I) The representative images of IF staining for DNA/NE/MPO in human neutrophils from healthy controls, HCC patients and HBV‐HCC patients. (J) ELISA analysis for serum levels of MPO‐DNA (serum marker for NETs) in healthy controls, HCC patients and HBV‐HCC patients. White scale bars: 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: HCC, HBV‐negative hepatocellular carcinoma; HBV‐HCC, HBV‐positive hepatocellular carcinoma; IOD, integrated optical density; DAPI, 4’, 6‐diamidino‐2‐phenylindole; CitH3, citrullinated modification of histone 3; NE, neutrophil elastase; MPO, myeloperoxidase; IF, immunofluorescence; N, normal; C, cancer; HC, healthy controls.