Journal: Frontiers in Cellular and Infection Microbiology

Article Title: MiR-199a-3p-regulated alveolar macrophage-derived secretory autophagosomes exacerbate lipopolysaccharide-induced acute respiratory distress syndrome

doi: 10.3389/fcimb.2022.1061790

Figure Lengend Snippet: MiR-199a-3p mimics augmented LPS-induced SAP secretion via the activation of Rab8a in vitro . (A) Western blot analysis demonstrated the Rab8a levels in RAW264.7 cells with the transfection of MiR-199a-3p mimics or inhibitors. (B) Western blot analysis illustrated the efficient siRNA-mediated knockdown of Rab8a in RAW264.7 cells with the transfection of MiR-199a-3p mimics or inhibitors. (C) NTA revealed significant decreases in vesicle levels from RAW264.7 cells with Rab8a knockdown in the MiR-199a-3p mimic and inhibitor groups compared with the control group. (D) Western blot analysis demonstrated significant decreases in LC3-II levels in vesicles from LPS-stimulated RAW264.7 cells in the Rab8a knockdown groups compared with the control group. The experiments were repeated at least three times (n = 3 per group). Each value represents the mean ± SD of three independent experiments. *p < 0.05 vs . the control; #p < 0.05 vs . the LPS control; **p < 0.05 vs . the mimics group without Rab8a knockdown; ***p < 0.05 vs . the inhibitors group without Rab8a knockdown, one-way ANOVA.

Article Snippet: Then, we blocked the membranes with 5% bovine serum albumin (BSA) for 1 h at room temperature and hatched with primary antibodies overnight at 4°C, which included antibodies against F4/80, LC3B, β-actin, Rab8a (1:1000; #6975, #43566,#4970 and #30325, CST), and PAK4 (1:1000, pS474, Absin).

Techniques: Activation Assay, In Vitro, Western Blot, Transfection, Knockdown, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: MiR-199a-3p-regulated alveolar macrophage-derived secretory autophagosomes exacerbate lipopolysaccharide-induced acute respiratory distress syndrome

doi: 10.3389/fcimb.2022.1061790

Figure Lengend Snippet: MiR-199a-3p mimics activate Rab8a by directly reducing PAK4 expression. (A) The predicted MiR-199a-3p binding site on the PAK4 3′-UTRs was determined using target prediction software. (B) Western blot analysis validated the PAK4 expression when cells were transfected with the MiR-199a-3p mimic/inhibitor. (C) Luciferase reporter plasmid assays with wild-type and mutated PAK4 plasmids co-transfected with MiR-199a-3p- or MiR-NC-packaged plasmids. Dual luciferase control vector plasmids acted as NCs. (D) Nanoparticle tracking analysis revealed significant increases in the level of vesicles from PF-3758309-stimulated RAW264.7 cells in the MiR-199a-3p mimic and inhibitor groups compared with the control group. (E) Western blot analysis demonstrated significant increases in LC3-II levels in vesicles from PF-3758309-stimulated RAW264.7 cells in the MiR-199a-3p mimic and inhibitor groups compared with the control group. (F) Western blot analysis revealed the effect of PF-3758309 on increasing Rab8a levels in RAW264.7 cells transfected with MiR-199a-3p mimics and inhibitors. The experiments were repeated at least three times (n = 3 per group). Each value represents the mean ± SD of three independent experiments. *p < 0.05 vs . the plasmid-control group transfected with MiR-199a-3p plasmids; #p < 0.05 vs . the control group; **p < 0.05 vs . the mimics group without PF-3758309; ***p < 0.05 vs . the inhibitors group without PF-3758309, one-way ANOVA.

Article Snippet: Then, we blocked the membranes with 5% bovine serum albumin (BSA) for 1 h at room temperature and hatched with primary antibodies overnight at 4°C, which included antibodies against F4/80, LC3B, β-actin, Rab8a (1:1000; #6975, #43566,#4970 and #30325, CST), and PAK4 (1:1000, pS474, Absin).

Techniques: Expressing, Binding Assay, Software, Western Blot, Transfection, Luciferase, Plasmid Preparation, Control