Fig 2: Interferon-ß (IFN-ß) affects the expression of macrophage polarization-specific cytokines. (A) RT-qPCR analysis of the silencing efficiency of siIFNB1, **P<0.01. (B) ELISA of the silencing efficiency of siIFNB1-3 or neutralizing efficiency of anti-IFN-ß, *P<0.05. (C and D) RT-qPCR analysis of mRNA expression of IL-12p40, IL-12p35, IL-23p19, IL-6, TNF-a, and IL-10 in M1 macrophages transfected with siIFNB1-3 (siIFNB1) or siC, or neutralized with anti-IFN-ß antibody (Ab), *P< 0.05, **P<0.01. (E) Histograms showing surface staining for CD86 and CD206 expression on siIFNB1- or siC-transfected U937-M1 cells. Data were calculated from 3 independent experiments. (F) The raw flow cytometry fluorescence data are representative of 3 independent experiments for CD86 and CD206 expression on U937-M1 transfected with siIFNB1 or siC. The percentage of positive cells and mean fluorescence intensities (MFIs) are denoted. (G and H) ELISA of the IL-12p70 and IL-10 secretion by M1 macrophages transfected with siIFNB1 or siC or neutralized with anti-IFN-ß antibody. Data were calculated from 3 independent experiments. *P<0.05, **P<0.01; n.d, not detected.

Fig 3: Interferon regulatory factor 1 (IRF1) regulates the expression of macrophage polarization–specific cytokines. (A) RT-qPCR analysis of the silencing efficiency of 3 siRNAs targeting IRF1, **P<0.01. Western blot analysis of the silencing efficiency of siIRF1-2, *P<0.05. The western blot analysis chart is a representative of 3 independent experiments. (B) RT-qPCR analysis of the mRNA expression of IL-12p40, IL-12p35, IL-23p19, IL-6, TNF-a and IL-10 in M1 macrophages transfected with siIRF1-2 (siIRF1) or the control siRNA (siC), **P<0.01. (C) ELISA of IL-12p70 and IL-10 secretion in M1 macrophages transfected with siIRF1-2 (siIRF1) or siC, *P<0.05, **P<0.01; n.d, not detected. (D) Histograms showing surface staining for CD86 and CD206 expression on siIRF1- or siC-transfected U937-M1 cells, *P<0.05. Data were calculated from 3 independent experiments. (E) The raw flow cytometry fluorescence data are representative of 3 independent experiments for CD86 and CD206 expression; staining profiles of U937-M1 cells transfected with siIRF1 or siC. The percentage of positive cells and mean fluorescence intensities (MFIs) are denoted.

Fig 4: Expression of cytokines and markers of U937-macrophages stimulated with interferon-? (IFN-?) and lipopolysaccharide (LPS). (A) RT-qPCR analysis of M1-associated genes, including IL-12p40, IL-12p35, IL-23p19, IL-6 and TNF-a, and the M2-associated gene, IL-10, in U937 unstimulated (only treated with 5 ng/ml PMA; labeled as PMA) or stimulated with IFN-? (20 ng/ml) and LPS (100 ng/ml) (labeled as IFN-? + LPS) for 8 h, **P<0.01. (B) ELISA of the IL-12p70 and IL-10 secretion levels in PMA or IFN-? + LPS group, *P<0.05. (C) The raw flow cytometry fluorescence data are representative of 3 independent experiments for CD86 and CD206 expression; staining profiles of PMA or IFN-? + LPS-stimulated U937 cells. The percentage of positive cells and mean fluorescence intensities (MFIs) are denoted. (D) Histograms shows surface staining for CD86 and CD206 expression on PMA or IFN-? + LPS-stimulated U937 cells, **P<0.001. Data were calculated from 3 independent experiments.

Fig 5: miRNA function in human B cell.(a) Inhibition of miRNA biogenesis affects miRNA function. Depletion of Dicer, Ago2, or TRBP was accomplished by specific shRNA. The influence on miRNA function was calculated by luciferase reporter with miR-200c, miR-203, and miR-31 target sequences in 3'UTR (n = 3). Each knockdown decreased endogenous miRNA function and increased Luciferase activity in 293T cells. Up to 80% of knockdown efficiency was confirmed by qRT-PCR (data not shown). (b,c) Inhibition of miRNA biogenesis affects proliferation of human primary B cells induced by anti-IgM (5 µg/ml). Dynamic proliferation was examined for 3 days in CD19 + B cells with shRNA targeting RISC components. The shRNA expression was monitored by Venus expression. Representative results (n = 3) of Venus/proliferation (b) and time-course (c) are shown. Cell proliferation was estimated by CellVue Claret Far Red Fluorescence. Reciprocal relationship between the fluorescence and cell numbers was confirmed (Supplementary Fig. 1a). (d) Surface CD86 expression in each shRNA-expressing populations (CD19+/Venus +) under the treatment of anti-IgM (0.1 and 5 µg/ml). (e) Inhibition of miRNA biogenesis affects B cell proliferation induced by various stimuli (anti-IgM, 5 µg/ml; BAFF, 0.2 µg/ml; anti-CD40, 2 µg/ml, IL-4, 20 ng/ml). Dynamic proliferation was examined for 3 days in CD19+B cells with shAgo2 (solid lines) and shCtrl (dashed lines). (f) Knockdown of Ago2 affects signaling activities in primary B cells assessed by western blotting. Lentivirus transduction was confirmed by Venus expression.