Fig 2: PRTN3 mRNA and protein levels in GC tissue samples. (A,B) PRTN3 mRNA and protein levels in 20 paired GC and paracancerous tissues was detected via qRT-PCR and Western blotting. C and N respectively denote cancerous and noncancerous tissues. (C) Immunohistochemical analysis of PRTN3 levels in 47 paired GC and paracancerous tissues in the tissue microarray. (D) Representative images of the levels of PRTN3 in two paired samples of GC and paracancerous tissue (left scale bar: 500 µm; right scale bar: 100 µm). Staining was done using hematoxylin reagent and observation was done with fluorescence microscope. Data are the mean ± standard deviation. *, P<0.05; **, P<0.01; ***, P<0.001. GC, gastric cancer; PRTN3, proteinase 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction.

Fig 3: PRTN3 silencing suppressed GC cell proliferation. (A) PRTN3 levels in GC cells were evaluated via qRT-PCR and Western blotting. (B,C) Efficiency of PRTN3 knockdown in lentivirus-transduced AGS cells and BGC-823 cells as assessed by qRT-PCR and Western blotting. (C) CCK-8 and (D) colony formation (were used to assess cell proliferation after transduction with the indicated constructs). Staining was done using crystal violet stain and observation was done using a microscope. Data are the mean ± standard deviation. *, P<0.05; **, P<0.01. PRTN3, proteinase 3; mRNA, messenger RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; shNC, negative control short hairpin RNA; OD, optical density; GC, gastric cancer; qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; CCK-8, Cell Counting Kit-8.

Fig 4: PRTN3 silencing inhibited in vivo GC tumor growth. (A,B) Tumor size, weight, and volume were quantified for AGS cells and BGC-823 cells derived from control cells or cells in which PRTN3 was knocked down when transplanted in nude mice. (C,D) PRTN3 mRNA and protein levels in tumors from the indicated mice. Data are the mean ± standard deviation. *, P<0.05; **, P<0.01; ***, P<0.001. shNC, negative control short hairpin RNA; PRTN3, proteinase 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; GC, gastric cancer.

Fig 5: Immunofluorescence detection of NET formation. A Levels of NET-related molecules (MPO, PAD4, PR3, NE and CitH3) were visualized and evaluated by TissueFAXS Spectra Multispectral Cytometers, and areas of colocalization (merged) are shown. Blue: DAPI; B Bar chart of average fluorescence intensity. The data represent the mean ± SEM; ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.