Journal: Frontiers in immunology

Article Title: Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS.

doi: 10.3389/fimmu.2018.00211

Figure Lengend Snippet: Figure 2 | Beta-defensins 2 (BD2) and beta-defensins 3 (BD3) suppressed autophagy in macrophages. (A–C) THP-1 macrophages were treated with recombinant human BD2 or BD3 or both peptides (1 µg/ml) for 6 h, and infected with Pseudomonas aeruginosa (PA) at multiplicity of infection 1 for 6 h. (D–G) RAW264.7 cells were transiently transfected with siBD2, siBD3, or both vs siNC for 24 h, and then infected with PA. (A,F) Cells were fixed, stained with anti- microtubule associated protein 1 light chain 3 (LC3) Alexa Fluro 488 fluorescent Ab, and then detected by immune fluorescence microscopy. Arrows indicate the LC3 puncta in THP-1 macrophages (A) and RAW264.7 cells (F). (B,G) Quantification of cells containing LC3 puncta in THP-1 macrophages (B) and RAW264.7 cells (G). (C,E) Protein levels of LC3-II were tested by western blot in THP-1 macrophages (C) and RAW264.7 cells (E). (D) The knockdown efficiency was tested by RT PCR. Data are shown as the mean ± SEM of three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Primary antibody against microtubule associated protein 1 light chain 3 (LC3) (REF NB910-40435, Novus Biologicals, Littleton, CO, USA), anti-β-actin (REF A1978, clone AC-15, Sigma-Aldrich, St. Louis, MO, USA), anti-Lamin B (REF sc-6216, clone B-10, Santa Cruz Biotechnology, CA, USA), anti-human betaDefensin 3 (REF AE1050, clone L3-18b-E1, Immundiagnostik), anti-human beta-Defensin 2 (REF AE1110, clone L12-4C-C2, Immundiagnostik, Produktion, Germany), anti-c-FOS (REF sc-52, clone E-4, Santa Cruz Biotechnology, CA, USA), and antiEGR1 (REF sc-189, clone C-19, Santa Cruz Biotechnology, CA, USA) were used in this study.

Techniques: Recombinant, Infection, Transfection, Staining, Fluorescence, Microscopy, Western Blot, Knockdown, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in immunology

Article Title: Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS.

doi: 10.3389/fimmu.2018.00211

Figure Lengend Snippet: Figure 5 | Beta-defensins 2 (BD2) and beta-defensins 3 (BD3) promoted macrophage-mediated phagocytosis and intracellular killing of Pseudomonas aeruginosa (PA) by inhibiting autophagy. RAW264.7 cells were transfected with siBD2, siBD3, siBecin1, or siATG7 for 24 h, and infected with PA at multiplicity of infection 25 for 1 or 2 h. Microtubule-associated protein 1 light chain 3 (LC3)-II protein expression was determined by western blot (A,D). Phagocytosis (B,E) and intracellular killing (C,F) was accessed by plate count assay. Data are shown as the mean ± SEM of three independent experiments (**p < 0.01; ***p < 0.001).

Article Snippet: Primary antibody against microtubule associated protein 1 light chain 3 (LC3) (REF NB910-40435, Novus Biologicals, Littleton, CO, USA), anti-β-actin (REF A1978, clone AC-15, Sigma-Aldrich, St. Louis, MO, USA), anti-Lamin B (REF sc-6216, clone B-10, Santa Cruz Biotechnology, CA, USA), anti-human betaDefensin 3 (REF AE1050, clone L3-18b-E1, Immundiagnostik), anti-human beta-Defensin 2 (REF AE1110, clone L12-4C-C2, Immundiagnostik, Produktion, Germany), anti-c-FOS (REF sc-52, clone E-4, Santa Cruz Biotechnology, CA, USA), and antiEGR1 (REF sc-189, clone C-19, Santa Cruz Biotechnology, CA, USA) were used in this study.

Techniques: Transfection, Infection, Expressing, Western Blot

Journal: Frontiers in immunology

Article Title: Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS.

doi: 10.3389/fimmu.2018.00211

Figure Lengend Snippet: Figure 7 | Early growth response gene-1 (EGR1) or c-FOS enhanced autophagy in macrophages. (A–H) THP-1 macrophages were transfected with specific siRNA or overexpression plasmid for EGR1 or c-FOS for 24 h, and then infected with Pseudomonas aeruginosa (PA) for 1 h. Knockdown and overexpression effects of EGR1 (A,E) and c-FOS (B,F) were determined by western blot. Protein levels of microtubule associated protein 1 light chain 3 (LC3)-II (C,D,G,H) were tested by western blot in THP-1 macrophages before or after PA infection.

Article Snippet: Primary antibody against microtubule associated protein 1 light chain 3 (LC3) (REF NB910-40435, Novus Biologicals, Littleton, CO, USA), anti-β-actin (REF A1978, clone AC-15, Sigma-Aldrich, St. Louis, MO, USA), anti-Lamin B (REF sc-6216, clone B-10, Santa Cruz Biotechnology, CA, USA), anti-human betaDefensin 3 (REF AE1050, clone L3-18b-E1, Immundiagnostik), anti-human beta-Defensin 2 (REF AE1110, clone L12-4C-C2, Immundiagnostik, Produktion, Germany), anti-c-FOS (REF sc-52, clone E-4, Santa Cruz Biotechnology, CA, USA), and antiEGR1 (REF sc-189, clone C-19, Santa Cruz Biotechnology, CA, USA) were used in this study.

Techniques: Transfection, Over Expression, Plasmid Preparation, Infection, Knockdown, Western Blot