Fig. 5. t-NETs induce CAF activation.

A Representative plot illustrating growth of CAFs in the presence of vehicle or micrococcal nuclease detached NETs. B Phase contrast images and respective quantification of the percentage area of cell free space per field after treatment of pancreatic FBs or CAFs with NETs derived from CAF CMed treated neutrophils for 24 h. (C) Quantification of the size of collagen gels 24, 48, and 72 h after seeding pancreatic CAFs treated with or without NETs derived from CAF CMed treated neutrophils for 24 h. D Expression of Acta2 and Col1a2 at the gene level in pancreatic CAFs treated with or without NETs derived from CAF CMed treated neutrophils for 24 h. E Confocal microscopy of Collagen1 and phalloidin on pancreatic CAFs treated with or without NETs derived from unstimulated neutrophils or CAF CMed treated neutrophils for 24 h. F Cellularity of melanoma after treatment with GSK484, Cl-amidine, BACEi or vehicle expressed as cells per unit volume. G Representative confocal image of nuclei and collagen after treatment with GSK484, Cl-amidine, or vehicle. Tissues stained with Herovici stain to demark new collagen (blue) vs. mature collagen (pink). H Representative plot illustrating growth of CAFs following treatment with CAF CMed or CAF CMed and PAD4i-treated neutrophil-derived media. Bar graphs are mean ± SEM; *p < 0.05 and ***p < 0.001 using B–D a paired t-test and F a Mann–Whitney test. Box and whiskers graph-line: median, box: upper and lower quartiles, whiskers: maxima and minima. Assays were performed on (A and H) Representative of n = 2 (in duplicate), B n = 3 (in triplicate), C n = 3 (in duplicate or quadruple), D n = 4 (in duplicate), E Representative images of n = 3 samples, F n = 5–10 male and female, 8–24-wk-old mice, and G Representative images of n = 5 (Vehicle) and n = 5 (GSK484 and Cl-amidine) independent experiments. Scale bars are 50 µm (B and E), 500 µm (G upper panel) and 100 µm (G upper panel).