Figure 6
NET-associated miRNA-142-3p regulates PKCα expression in macrophages. (A,B) PMA-differentiated THP-1 macrophages were exposed to PMA-induced NET incubated or not with specific TYE-665-labelled hsa-miRNA-142-3p inhibitor or scramble sequence control, or treated with cytochalasin D (CytoD) before NET exposure. Salmon sperm DNA (salDNA) was used as control. After 2 h, cells were washed and complete culture medium was added. The expression of PKCα was evaluated using flow cytometry, showing positive population (A) and median fluorescence intensity ratio (MFI ratio, and B) 18 h after treatment, with (C) as a representative histogram. (D,E) THP-1 macrophages were exposed to NET associated or not with specific hsa-miRNA-142-3p inhibitor or scramble sequence control, or treated with salmon sperm DNA, or a PKC inhibitor (iPKC, Go6383, 50 nM). Cells were also treated with LPS (20 ng/ml) plus treatments above. After 2 h, cells were washed and complete culture medium was added. Culture supernatants were collected 6 h later and TNF-α production was quantified by ELISA. (E) Analysis of TNF-α production when normalized to levels induced by LPS. Results (A,C,D) derive from 4 independent experiments using one-way ANOVA, with Bonferroni post-hoc test, displaying mean ± standard error of mean of the test group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
