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. 2021 Nov 17;7(47):eabj0829. doi: 10.1126/sciadv.abj0829

Fig. 5. Skin ETosis is essential for preventing infection during terrestrialization in lungfish.

Fig. 5.

Terrestrialized lungfish (A) skin and (B) cocoon stained with DNA stain DAPI (blue), anti-H2A antibody (green), anti-human ELANE antibody (red), and merged. Cell suspensions (50,000 cells per treatment) were either not stimulated (control), stimulated with PMA, or stimulated with PMA and treated with the ETosis inhibitors diphenyleneiodium chloride (PMA + DC) or Cyto (PMA + Cyto). Cells were stained with DNA stain DAPI (blue), anti-H2A (green), anti-human ELANE antibody (red), and merged. (C) Representative confocal microscopy images of terrestrialized lungfish skin stimulated with PMA. (D) Representative confocal microscopy images of terrestrialized lungfish skin stimulated with PMA + Cyto. (E) Quantification of the percentage of cells undergoing ETosis in control terrestrialized lungfish skin (n = 2). PBS, phosphate-buffered saline. (F) Quantification of the percentage of cells undergoing ETosis in in vivo deoxyribonuclease I (DNase I)–treated terrestrialized lungfish skin (n = 2 fish). Counts were performed in n = 10 fields per fish and treatment. (G) Skin lesion and (H) prolapsed anus in DNase I–treated terrestrialized lungfish. (I) Giemsa-stained blood smear of terrestrialized lungfish treated with DNase I showing bacteria in circulation. (J) Average number of granulocytes in blood smears of control terrestrialized and DNase I–treated terrestrialized lungfish (n = 3). (K) Representative H&E stain of DNase I–treated lungfish skin from a wound area showing hemorrhages. (L) DNase I–treated mucus cocoon of terrestrialized lungfish (n = 3). (M to P) Quantification of gene expression levels of antimicrobial peptide genes (defb1 to defb4) in control free-swimming lungfish skin (C), terrestrialized lungfish skin (TS), and DNase I–treated terrestrialized lungfish skin (DTS) (n = 3 to 5). Gene expression levels were normalized to the house-keeping gene pgk-1. Data were analyzed by unpaired Student’s t test. *P < 0.05.