Fig. 1

Overexpression of miR-190 in zebrafish neutrophils reduces cell motility and chemotaxis. (A) Schematic of the Tol2-LyzC-miR-Dendra2 plasmid, injected into wild-type AB zebrafish embryos to generate Tg(lyzC-miR-190-dendra2)pu38 and Tg(lyzC-vector-dendra2)pu7 using the Tol2 transposon system. (B) Schematic of the zebrafish tail wounding assay showing Sudan black staining of wounded zebrafish to count neutrophils migrating to the wound at specific times after wounding. (C, D) Representative images and quantification of neutrophil recruitment. (Scale bar, 100 μm) The assay included 3 biological repeats with 17-20 fish per group. Data are shown as mean ± SD, analyzed with the Mann-Whitney test. (E) Schematic of the LTB4 chemotaxis assay showing Sudan black staining of zebrafish to measure the percentage of neutrophils migrating from the CHT to the caudal fin within specific times after LTB4 exposure. (F, G) Representative images and quantification of neutrophil chemotaxis from the CHT to the caudal fin in vector and miR-190 OE larvae after 15 min of LTB4 exposure. (Scale bar, 100 μm) The assay included 3 biological repeats with 20 fish per group. Data are presented as mean ± SD and analyzed using the Mann-Whitney test. (H, I) Representative images and quantification of live neutrophil motility in vector and miR-190 OE larvae over 30 min. (Scale bar, 100 μm). The experiment included three biological repeats with 22-39 neutrophils tracked from 1 to 4 fish per group. Data are presented as mean ± SD, and analyzed using the Mann-Whitney test. (J, K) GFP images and Sudan black staining quantifying whole-body neutrophils in vector and miR-190 OE larvae. (Scale bar, 100 μm) The assay consisted of three biological repeats, with 20 fish per group. Data are shown as mean ± SD, analyzed with the Mann-Whitney test. (L) Schematic of infection of the common cardinal vein with P. aeruginosa. (M) Survival analysis comparing miR-190 OE with vector controls. The experiment was repeated 3 times with a total of 95 miR-190 OE larvae and 91 vector control larvae. Data were analyzed using the Mantel-Cox test.