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. 2020 Feb 17;10:2715. doi: 10.1038/s41598-020-59486-2

Figure 3.

Figure 3

NET-associated miRNAs (NET-miRs) present differential patterns upon distinct types of stimuli. We constructed a miRNA expression profile for 87 different Taqman assays obtained with quantitative PCR from NET supernatants obtained with three activators (PMA, A25T, La). (A) Venn diagram (center) showing the number of amplified miRNAs for each stimulus; the number zero corresponds to non-detected miRNAs. The left graphic depicts an example of a miRNA that was amplified with PMA and A25T treatment, and the right graphic shows hsa-miR-432-5p, which was amplified only in the PMA and La samples. (B) Profiling of the expression values of 39 miRNAs from Leishmania (La, gray box), amyloid fibrils (A25T, black box) and PMA (white box) stimulated neutrophil supernatants. The color scale shown on the right illustrates the expression from the normalized data, in which red indicates an expression level higher than the mean across all subjects, gray denotes an expression level lower than the mean, and white indicates the median expression. Hierarchical cluster analysis subdivided the samples into three main groups, in which A25T and PMA treatments are clustered together in the dendrogram at the top of the heatmap, while the dendrogram on the left illustrates the miRNA clustering. (C) Bar plots showing the miRNA expression difference between the activators, displaying the mean expression levels ± SEM of the test groups (La and A25T) relative to the control group (PMA). To avoid donor-to-donor variation, we performed donor-paired analysis for the three NET activators. Statistical significance was calculated by comparison of the means of the normalized gene expression values between groups performed by a nonparametric one-way ANOVA with 1,000 unrestricted permutations, followed by post-hoc pair-wise comparisons with Bonferroni adjustment using a nonparametric t-test also with 1,000 permutations (**p < 0.001) and (*p < 0.05). Results are from 6 independent biological replicates for each activator. PMA, phorbol myristate acetate; A25T, amyloid fibrils; La, L. amazonensis promastigotes.