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. 2019 Jan 22;9:3111. doi: 10.3389/fimmu.2018.03111

Figure 6.

Figure 6

miRNA-mediated GILZ downregulation. (A) miRNA induction in Poly(I:C)-treated AMs (10 μg/mL for 2 h or 1 μg/mL for 24 h) was assessed by microarray analysis. Data are presented as fold change compared with untreated cells from the same donor. Individual changes per donor (n = 3) are shown. (B) The interaction of individual miRNAs with GILZ was determined by miRNA overexpression in HEKT293 cells co-transfected with a luciferase reporter construct containing the GILZ 3′UTR. Luminescence was measured 24 h after transfection and is shown as percent change compared with control vector-transfected cells lacking miRNA overexpression (n = 3–8, quintuplicates or sextuplicates). Significances were calculated in comparison with control vector-transfected cells. (C,D) HEK293T cells were transfected with miRNA mimics or scrambled controls (50 nM). GILZ expression was determined by Western blot at the indicated time points. (C) Representative blots. (D) GILZ signal intensities were quantified and normalized to tubulin values (n = 3, duplicates; Co: cells transfected with scrambled controls, set as 100%). (E,F) HEK293T cells were transfected with scrambled controls (50 nM, Co), miR-34b* (12.5 nM miR-34b* mimic + 37.5 nM scrambled controls), or a mix of miR-34b*, −222, −320d, and −484 mimics (12.5 nM each). GILZ expression was quantified after 48 h by Western blot. (E) Representative blot. (F) GILZ signal intensities were normalized to tubulin values (n = 3, duplicates or triplicates). Co values were set as 100%. (G) NF-kB/AP-1 activity was measured in untreated HEK-Blue reporter cells (Co) or after activation with PIC (1 μg/mL, 24 h) in the presence or absence of a cell-permeable GILZ peptide (TAT-GILZ, 2 μg/mL) or the respective control peptide (TAT-Co, 2 μg/mL; n = 3, triplicates). (H) HEK-Blue reporter cells were transfected with scrambled control (Co) or the indicated miRNA mimic. Twenty hours after transfection, cells were either left untreated or treated with PIC (1 μg/mL, 24 h), and NF-kB/AP-1 activity was determined (n = 3, triplicates). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Co or as indicated.