Figure 7. AP-1 and NF-κB1 Contribute to SPM-miR-466l Regulatory Networks.

(A) Human macrophages were transfected with VC, miR-466l, anti-miRNA-neg,oranti-miR-466lfor72 hr. Total proteins were isolated for IκBα, p-NF-κB, p-JNK, and p-p38 ELISA (miR-466l overexpression [OE]; miR-466l silencing [Si]). Results are normalized against VC or anti-miRNA-neg and expressed as mean ± SEM from four independent experiments. *p < 0.05, versus VC; ^#p < 0.05, versus anti-miRNA-neg.

(B and C) Human macrophages were exposed to Zym (10 ng/ml) and/or SPM (RvD1, RvD2, or MaR1 [10 nM each]) (37°C, 18 hr, pH 7.45). (B) Protein expression of p-JNK, p-p38, IκBα, and NF-κB, and (C) mRNA values of c-Jun and NF-κB1 were assessed with ELISA and qPCR, respectively. Results are expressed as mean ± SEM from five independent experiments. *p < 0.05 and **p < 0.01, versus Veh control; ^#p < 0.05 and ^##p < 0.01, versus Zym alone.

(D) Human macrophages were transfected with shAP-1, shNF-κB1, or shscrambled (sh-neg) for 48 hr. Expression levels of miRNA-biogenesis-associated genes, pri-miR-466l, pre-miR-466l, miR-466l, and select resolution-related miRNAs were determined with qPCR. Heatmap results were normalized with sh-neg as controls and are expressed as the mean of five independent experiments.

(E) Human macrophages were preincubated with PTX(100 ng/ml) for 24 hr (37°C, pH 7.45) and then treated with RvD1, RvD2, or MaR1 (10 nM each) for 18 hr (37°C, pH 7.45); expression levels of miR-466l, c-Jun, NF-κB1, TNFα, COX-2, and TLR4 were assessed by qPCR. Heatmap results were normalized against VC and are expressed as the mean of five independent experiments. The results of NF-κB1 and TNFα are expressed as mean ± SEM. *p < 0.05 and **p < 0.01, versus Veh control; ^#p < 0.05, versus RvD1 Veg; *p < 0.05, versus RvD2 Veg; ^‡p < 0.05 and ^‡‡p < 0.01, versus MaR1 Veg.

See also Figure S6.