miR-146b-5p Plays a Critical Role in the Regulation of Autophagy in ∆per Brucella melitensis-Infected RAW264.7 Cells

Figure 5

mmu-miR-146b-5p inhibits the autophagy activation in RAW264.7 cells during B. melitensis infection. (a) mmu-miR-146b-5p expression levels in RAW264.7 cells transfected with miR-146b-5p mimic were examined by qRT-PCR at 12 h, 24 h, and 36 h after transfection. (b) mmu-miR-146b-5p expression levels in RAW264.7 cells transfected with miR-146b-5p inhibitor were examined by qRT-PCR at 24 h after transfection, and the expression level of miR-146b-5p was downregulated significantly by the miR-146b-5p inhibitor. (c) RAW264.7 cells were seeded at 0 h, were transfected with miR-NC mimic or miR-146b mimic after 24 h, and were infected with B. melitensis M5-90 after 44 h, and the total protein was extracted after 48 h. (d) Total cell lysates in C were then prepared and subjected to immunoblot analysis using monoclonal anti-LC3-I antibody and polyclonal antibody against LC3-II. (e) The quantification of LC3-II/GAPDH levels in D with BandScan5.0 (n = 3). (f) RAW264.7 cells were seeded at 0 h, were transfected with miR-NC inhibitor or miR-146b inhibitor after 24 h, and were infected with B. melitensis M5-90 after 44 h, and the total protein was extracted after 48 h. (g) Total cell lysates in F were then prepared and subjected to immunoblot analysis using monoclonal anti-LC3-I antibody and polyclonal antibody against LC3-II. (h) The quantification of LC3-II/GAPDH levels in G with BandScan5.0 (n = 3). Data are mean ± SD from three independent experiments. ; .

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