Figure 5.

Increased stability of MCPIP-1 in neutrophils during apoptosis depends on miRNA depletion. (A) Freshly isolated human PMNs were preincubated for 1 h with 5 μg/ml of actinomycin D, then RNA was isolated and MCPIP-1 determined using qPCR. Data represent the relative expression of MCPIP-1 mRNA from one (out of 3) representative donor ± SEM. The level of MCPIP-1 directly post PMNs isolation was taken as 1. C, control; Act, actinomycin D; (B) Identification of miRNAs binding sites in the MCPIP-1 3′UTR transcript. (C) The mean expression of 6 sequences of miRNA was determined using qPCR. ± SD, n = 3–4; (D) Binding 200 nM Atto488-labeled MCPIP-1 oligonucleotide with 200 nM Cy5-labeled miR101-3p sequence was performed using EMSA assay. A representative result was presented. (*)-MCPIP-1 mRNA without dye (E) After indicated time post PMNs isolation from human blood the level of the miRNA expression was determined. Data represent results derived from a representative donor. Bars represent the relative miRNA expression to time 0 (t₀ = 1) ± SEM. (F) Neutrophils were exposed for 2 h to GM-CSF previously incubated or not for 15 min with neutralizing anti-GM-CSF antibodies. Control cells were treated with PBS or antibodies alone. qPCR analysis was performed for the estimation of miRNA expression relative to PBS treated cells equal 1. A representative result out of 3 was presented ± SEM. p-value of < 0.05 (*); p-value of < 0.01 (**); p-value of < 0.001 (***); ns for non-significant.