Fig. 4

MiR-125b-5p mediates intracellular B. abortus replication via the suppression of macrophages activation. a RAW264.7 cells transduced with Scr-miR or Pre-miR-125b-5p were infected with B. abortus for the indicated time periods. B. abortus intracellular growth was determined by CFUs assay. The group containing Scr-miR was defined as control. *P < 0.05 vs control. Data are expressed as mean ± SEM. b RAW264.7 cells, transduced with Scr-miR or Pre-miR-125b-5p, were infected with B. abortus for 36 h, immunofluorescence analysis of the infected cells was performed. The number of Brucella in 100 fields is shown under the confocal microscopy images. Green: Brucella; blue: cell nucleus; red: calnexin (scale bar = 20 um). c BMDMs transduced with Scr-miR or Pre-miR-125b-5p was infected with B. abortus for the indicated time periods. B. abortus intracellular growth was determined by the CFUs assay. The group containing Scr-miR was defined as control. *P < 0.05 vs control. Data are expressed as mean ± SEM. In the middle panel of c, BMDMs transduced with lentivirus containing scrambled microRNA (Scr-miR) or pre-miR-125b-5p, were treated with ELPS (1000 ng/ml) for 1 h. Cell lysates were subjected to immunoblotting analysis of A20. d, e and f RAW264.7 cells, transfected with Scr-miR or Pre-miR-125b-5p, were infected with B. abortus for 8, 16 and 24 h, respectively, TNFα (d) or NO production in the supernatants of cells was measured by ELISA (f), and iNOS protein expression in the cells was determined by immunoblotting with iNOS antibody (e) g and h RAW264.7 cells, transfected with Scr-miR or Pre-miR-125b-5p, were stimulated with 10 μg/ml Brucella LPS (BrLPS) or 1000 ng/ml ELPS for the indicated time (min). pERK (g) or IkBα ( h) expression was determined using antibodies specific for pERK or IkBα. Data are representative of three independent experiments. Quantitative data for IkBα, pERK and iNOS expression are shown under the Western blotting data. The maximal ratio of IkBα, pERK or iNOS expression /β-actin in the group was defined as 100 % (or control). *P < 0.05 VS control. NS indicates no significance