(A) Fluorescent microphotographs showing ecDNA aggregates stained with propidium iodide in human biliary sludge (HBS). Arrows indicate single swollen nuclei. 4 representative microphotographs from 6 patients. Scale bar, 100 μm.

(B) Size distribution of ecDNA aggregates in HBS. n = 6 patients.

(C) Neutrophil elastase (NE) enzymatic activity of aggregates from HBS. 6 patients in technical triplicates. Please see also Figure S1 A.

(D) Composite macrophotograph of human gallstones, previously immersed in an aqueous propidium iodide solution, under oblique white light (left) and 488 nm light (right) illumination, respectively; ecDNA deposits appear in red color. Shown is 1 out of 3 independent experiments. Scale bar, 1 cm.

(E) Squash preparations of human gallstones stained for ecDNA (red). Arrows indicate single swollen nuclei. Please see also Figure S1 B. Shown are 3 representative microphtographs out of 3 independent experiments. Scale bar, 100 μm.

(F) Neutrophil elastase (NE; green) or citrullinated histone H3 (citH3; green) stainings. Please see also Figure S1 C. Representative microphotographs from 3 independent experiments. Scale bar, 200 μm.

(G) Percent calcification of human gallstones assessed by micro-computed tomography (μCT). n = 72 gallstones. Dashed line indicates detection limit.

(H) 4 out of 72 representaive μCT scans of human gallstones revealing various calcification patterns (green); red lines: circumference of gallstone.

(I) Frequencies of type of calcification patterns. n = 72 gallstones. Scale bar, 10 mm; lower right, 20 mm. Please see also Figures 1 D and 1E.

(J) NE enzymatic activity kinetics of human gallstone material (n = 20) obtained from the surface, the body or the center of the stone (red line indicates 1 unit/g NE).