idase for 30 min at 37uC. Immunoreactivity was visualized with 0.05% diaminobenzidine . Negative controls received the same treatments except that primary antibodies were omitted, and showed no specific staining. For double fluorescence immunostaining of Tra2b and RGS4, sections were incubated with goat anti-Tra2b antibody and rabbit anti-RGS4 antibody at 4uC, overnight. Sections were then incubated with Alexa Fluor -conjugated secondary antibodies at 37uC for 2 hours to reveal the positive signals. 
The specificity of antibodies in immunofluorescence was confirmed by the blocking experiments shown in Fig. S3. For MedChemExpress SB366791 triple staining of Tra2b, RGS4 and TH, sections were firstly incubated with goat anti-Tra2b antibody overnight and then with the corresponding Alexa Fluor -conjugated anti-goat secondary antibody. After rinsing in PBS, the sections were incubated with rabbit anti-RGS4 antibody and mouse monoclonal anti-TH antibody overnight and then with corresponding Alexa Fluorconjugated donkey anti-rabbit and donkey anti-mouse secondary antibodies. After washing, sections with fluorescence immunolabeling were mounted on glass slides and coverslipped using fluoromount medium. Fluorescence was detected by confocal laserscanning microscopy. Autophagy Enhances Bacterial Clearance during P. aeruginosa Lung Infection Robert D. 26507655 Junkins1,2,3, Ann Shen1, Kirill Rosen4, Craig McCormick1,3, Tong-Jun Lin1,2,3 1 Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada, 2 Department of Pediatrics, IWK Health Centre, Halifax, Nova Scotia, Canada, 3 Beatrice Hunter Cancer Research Institute, Halifax, Nova Scotia, Canada, 4 Department of Biochemistry and Molecular Biology, Halifax, Nova Scotia, Canada Abstract Pseudomonas aeruginosa is an opportunistic bacterial pathogen which is the leading cause of morbidity and mortality among cystic fibrosis patients. Although P. aeruginosa is primarily considered an extacellular pathogen, recent reports have demonstrated that throughout the course of infection the bacterium acquires the ability to enter and reside within host cells. Normally intracellular pathogens are cleared through a process called autophagy which sequesters and degrades portions of the cytosol, including invading bacteria. However the role of autophagy in host defense against P. aeruginosa in vivo remains unknown. Understanding the role of autophagy during P. aeruginosa infection is of particular importance as mutations leading to cystic fibrosis have recently been shown to cause a blockade in the autophagy pathway, which could increase susceptibility to infection. Here we demonstrate that P. aeruginosa induces autophagy in mast cells, which have been recognized as sentinels in the host defense against bacterial infection. We further demonstrate that inhibition of autophagy through pharmacological 9533644 means or protein knockdown inhibits clearance of intracellular P. aeruginosa in vitro, while pharmacologic induction of autophagy significantly increased bacterial clearance. Finally we find that pharmacological manipulation of autophagy in vivo effectively regulates bacterial clearance of P. aeruginosa from the lung. Together our results demonstrate that autophagy is required for an effective immune response against P. aeruginosa infection in vivo, and suggest that pharmacological interventions targeting the autophagy pathway could have considerable therapeutic potential in the treatment of P. aeruginosa lung infec