under G418 selection. HMC-1 5C6 cells stably MedChemExpress Peretinoin expressing NT, Atg5 or Atg7 shRNA were generated by culturing nucleofected cells under 10 mg/mL puromycin selection for 4 weeks starting 24 hours after nucleofection. Knockdown was confirmed by Western blot analysis as described below. LC3-GFP assay Two million HMC-1 5C6 cells stably expressing LC3-GFP were left untreated or treated as indicated. Cells were then fixed at various time points in 4% paraformaldehyde and cytocentrifuged onto glass slides for 5 minutes at 200 rpm for examination by fluorescence microscopy and by confocal laser scanning microscopy. Alternatively 16HBE14o2 cells were grown to confluence on poly-L-lysine coated glass coverslips and transiently transfected with LC3-GFP using Lipofectamine 2000 according to the manufacturer’s instructions. Cells were then left untreated, or treated as indicated and fixed in 4% paraformaldehyde and examined as described above. At least one hundred cells per slide were 
counted and cells containing 5 or more GFP punctuations were considered LC3 positive. Mast cell tryptase solid phase ELISA Mice were left untreated, or infected with P. aeruginosa strain 8821 as described above. Lung tissue homogenates, BALF supernatants and serum proteins were absorbed onto a high binding Nunc MaxiSorp 96 well plate overnight at 4uC. Plates were washed 4 times with 200 mL 0.01% Tween-20 in PBS then blocked for 2 hours at room temperature in 2% BSA in PBS. Plates were washed 4 times with 200 mL 0.01% Tween-20 in PBS then anti-mouse tyrptase antibody diluted 1:50 in 0.2% BSA, 0.05% Tween-20 in PBS was added and incubated overnight at 4uC. Plates were washed 4 times with 200 mL 0.01% Tween-20 in PBS then biotin conjugated mouse anti-goat IgG diluted 1:100 in 0.2% BSA, 0.05% Tween-20 in PBS was added and incubated at room temperature for 2 hours. Plates were washed 5 times with 200 mL 0.01% Tween-20 in PBS then ELISAs were developed using the Invitrogen ELISA amplification system according to the manufacturer’s directions. Lung infection with P. aeruginosa and collection of lung and bronchioalveolar lavage fluid Mice were infected with 16109 CFU P. aeruginosa intranasally for 24 hours. BALF was then obtained by lavaging the lung with 36 1 mL PBS containing 100 mg/mL soybean trypsin inhibitor. 8901831 title=’View abstract’ target=’resource_window’>14557281 The lung tissues were obtained for detection of cytokines, myeloperoxidase and bacterial colony-forming units counting. Lung tissues were homogenized in 50 mM HEPES buffer containing 100 mg/mL soybean trypsin inhibitor. For counting bacterial CFU, 10 mL of the homogenate was plated onto an agar dish and incubated for 24 h at 37uC. The homogenate was centrifuged at 4uC for 30 min at 18000 g. The supernatant was stored at 280uC for later cytokine analysis. The pellet was resuspended and homogenized in 0.5% cetyltrimethylammonium chloride and centrifuged as above. The cleared extract was used for MPO assay. BALF was plated on an agar dish and incubated for 24 h for CFU counting. For detection of cytokines and MPO activity, BALF was centrifuged at 480 g for 5 minutes at 4uC. The supernatants were used for cytokine analysis. The pellets were resuspended in 1 mL NH4Cl and centrifuged as before to lyse red blood cells. The supernatants were discarded and the pellets were resuspended in 0.5% CTAC then centrifuged. The cleared extracts were used for MPO assay. For survival studies, mice were infected with 16109 CFU P. aeruginosa strain 8821 as described above. Mice were then mon
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