Earle s balanced salt solution

HAV stock was obtained from the American Type Culture Collection (Manassas, VA) as VR1402, a cell culture-adapted cytopathic clone of strain HM-175 that was originally designated as HM-175/18f. The virus was propagated on fetal rhesus monkey kidney (FRhK-4) cells as previously described [15] (link). The HAV stock was stored at −70°C in DMEM (Gibco, Grand Island NY) with 10% fetal bovine serum (FBS) (Gibco) prior to use. To partially purify HAV, the virus was pelleted at 490,000×g for 6 h, followed by resuspension of the HAV in human plasma (George King Biomedical Inc., Overland Park, KS) and filtration through a 0.1 µm filter. Samples of human plasma alone or human plasma spiked with HAV were laser-irradiated as described above. Plaque assay was performed by making an initial 100-fold dilution followed by 10-fold serial dilutions made in Earle’s balanced salt solution (Gibco) and infecting 100-mm dishes confluent with FRhK-4 cell and infecting with 2 ml of virus dilution as described previously [16] (link). After 2 h, the plates were overlaid with DMEM medium with 5% FBS, and 1% agarose (Sigma-Aldrich, St. Louis, MO). At 17 days post-inoculation, HAV was inactivated by 10% formaldehyde treatment, the agarose overlay was removed, and HAV plaques were visualized by crystal violet staining (Fisher Scientific, Kalamazoo, MI).

Lung lobes were inflated under controlled pressure via the bronchial system with 2% agarose, low-gelling temperature (Sigma-Aldrich, St. Louis, MO) in Dulbecco’s modified Eagle’s medium Nutrient Mixture F-12 Ham (DMEM/F-12 HEPES, no phenol red) from Gibco (Darmstadt, Germany). Agarose-filled lobes were cut into approximately 1-cm-thick slices and macroscopically evaluated by a pulmonary pathologist (C.W. under the supervision of D.D.J.) to exclude neoplasms and infections. Human PCLS (8 mm in diameter and 200 to 300 μm thick) were generated in cold Earle's Balanced Salt Solution (Gibco) using an Alabama RD MD6000 Tissue Slicer (Alabama Research and Development, Munford, AL) as described previously.¹² (link)^,13 (link)^,38 Two PCLS per well were incubated in 24-well culture plates (Supplemental Figure S1) with 500 μL of DMEM/F12 supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL) under standard cell culture conditions (37°C, 5% CO₂, and 100% humidity), and the medium was changed regularly. Before medium change, each well that contained PCLS was stereoscopically (eightfold to 40-fold magnification) assessed using a Zeiss (Oberkochen, Germany) stereoscope to identify bacterial or fungal contaminations.

ME180 cells were mock-infected with GCB medium alone or with Ngo, for the indicated times. Starvation, as a positive control for induction of autophagy, was performed by incubating cells in Earle’s Balanced Salt Solution (Gibco). To induce LC3-II accumulation, cells were incubated with chloroquine diphosphate (Sigma) or bafilomycin A1 (Sigma) for 1 h prior to infection and maintained in the medium throughout the experiment. To terminate infection, unattached bacteria were removed by washing the cultures twice with ice-cold PBS. Cells were then lysed with 120 μL of RIPA2 lysis buffer (150 mM NaCl, 5 mM EDTA pH 8.0, 50 mM Tris pH 8.0, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS). Lysates were mixed 1:1 with Tris-Tricine sample buffer (Bio-Rad, Hercules, California, USA) and 1 tablet of protease inhibitor cocktail (Roche, Indianapolis, Indiana, USA). The samples were boiled for 10 min, and then separated in a SDS 12% Tris-Tricine polyacrylamide gel containing urea (6M). The separated proteins were transferred to PVDF membranes (0.1 μm, GE, Fairfield, Connecticut, USA) and probed with the appropriate antibodies (overnight at 4°C).

Embryos (E10.5 pc) were dissected from the mouse decidua and extra embryonic tissues and pooled into Earle’s balanced salt solution (Gibco). Hearts were dissected from the embryos and the AV regions were removed, and cut to expose the lumen. The AV explants were placed immediately onto the surface of drained collagen gels with 1 explant per well, and incubated at 37°C for 3–4 hr to allow attachment of the explants onto the collagen gel surface as previously described [29] (link), [30] (link). A total of 0.5 ml of DMEM (5% fetal bovine serum, antibiotics) was added to each well and incubated for a total of 48 hr at 37°C. The number of mesenchymal cells was assessed by observing the living cultures with a Zeiss Axiovert 100 inverted microscope with Hoffman phase contrast optics and “optically sectioning” the collagen gel by changing focal planes to reveal the presence of any cells beneath the surface of the endothelial monolayer for counting. Total mesenchymal cell counts were made from each explant and then later grouped according to genotype as determined in separate PCR assays performed after the explant EMT experiments were scored. Statistical analysis was performed to determine if significant differences exist between the mean values obtained from wild-type and mutant using the unpaired (student’s) t-test within the Prism GraphPad analysis software.