tumor cells rat tail collagen 1  (Chem Impex International)

Journal: Nature microbiology

Article Title: Pseudomonas aeruginosa infection augments inflammation through miR-301b repression of c-Myb-mediated immune activation and infiltration

doi: 10.1038/nmicrobiol.2016.132

Figure Lengend Snippet: (a) MH-S and MLE-12 cells were transfected with Ctrl siRNA or c-Myb siRNA for 24 h and were treated with LPS (100 ng/ml) for 6 h. mRNA of c-Myb, IL-4, and TGF-β1 was measured by qRT-PCR. (b) c-Myb, IL-4, and TGF-β1 protein was assessed in MLE-12 cells from above by immunoblotting. (c) mRNA levels of IL-6 and TNF-α were determined in the MH-S and MLE-12 cells by qRT-PCR. (d) Mice (n=3) were i.v. injected with Ctrl siRNA or c-Myb siRNA (50 μg/mouse, 24 h) and were infected with Pa (1×10 7 CFU) for 24 h. Lungs were evaluated by H&E staining. Scale bar=100 μm. (e) Lungs were evaluated by immunostaining with a Ly6G antibody to show neutrophil infiltration. Scale bar=50 μm. (f, g) After treatment as above, MPO assay and ELISA were performed to determine myeloperoxidase activities in lung homogenates or MIP-1α and IL-17A levels in BAL. Panel a, c, means+SD from triplicates; b, representative from triplicate samples; d, e, representative of the 3 mice; f, g, means+SD from 3 mice. *, p <0.05, **, p <0.01. One-way ANOVA with Tukey’s post hoc.

Article Snippet: TLR4, MyD88, TRIF, PKA, p65, CREB-1 and c-Myb siRNA were bought from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). miRNA negative control (NS-m, CN-001000-01-05), miR-301a mimics (301a-m, C-310482-05), miR-301b mimics (301b-m, C-310775-03), miR-302b mimics (302b-m, C-310669-05) miRNA inhibitor negative control (NS-i, IN-001005-01), or miR-301b inhibitor (301b-i, IH-310775-04) were bought from Dharmacon Inc. (Chicago, IL).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Injection, Infection, Staining, Immunostaining, MPO Assay, Enzyme-linked Immunosorbent Assay