Figure 4

Figure 4 ZAKα and NLRP1 contribution to Portimine-induced skin inflammation in 3D skin and zebrafish models.

(A) Representative diagram indicating the experimental approach in the WT and ZAKα-deficient 3D skin model of Portimine exposure. (B) Hemalun (H) & Eosin (E) staining showing ZAKα-dependent histological changes caused by PortimineA (20 nM) exposure. Yellow arrows show epidermidis alterations/damages induced by Portimine A. Associated quantification of the dermal–epidermal layer detachment of 3D skin. P values indicated in figure, one-way ANOVA. Images are representative of three biological replicates. Scale bar = 50 µm. (C) IL-1α, β, and -18 cytokine analysis in 3D skin treated or not with Portimine A (A, B). Results are from a total of 65 cytokine analysis presented in Fig. EV4A. P values indicated in figure, one-way ANOVA. (D) Survival curves of zebrafish larvae (20 larvae/group) incubated with various concentrations of Portimine A. Graph shows a representative experiment out of the three performed. P values indicated in figure, Log-Rank (Mantel–Cox) test. (E) Determination of skin necrosis induced by Portimine A (25 nM) in the caudal fin of zebrafish larvae by measuring the incorporation of the plasma membrane impairment probe SYTOX Green after 24 h exposure in area delimited by dashed white line. In total, 20–30 larvae/group were compared and quantified. Values are expressed as mean. P values indicated in figure, one-way ANOVA. Scale bar 100 µm. Graphs show one experiment performed three times. (F) Determination and quantification of zebrafish larvae fin tail gross damage induced by Portimine A (25 nM) in WT and Nlrp1- and ZAKa-deficient larvae (20/group) at the indicated time points. Scale bar 100 µm. Specific quantifications and statistical analysis are provided in experiments that measure tail area presented Fig. EV4B,D. Graphs show one experiment performed two times. (G) Determination and quantification of zebrafish neutrophil recruitment to the fin tail in WT and Nlrp1- and ZAKa-deficient larvae (20/group) of the zebrafish line Tg(LysC:GFP)^nz117 with GFP-expressing neutrophils over 24 h exposure to Portimine A (75 ng/mL). Graph shows a representative experiment out of the two performed. Values are expressed as mean. P values indicated in figure, one-way ANOVA (Kruskal–Wallis test). Scale bar 100 µm. Graphs show one experiment performed two times. Source data are available online for this figure.