Streptomycin

Tracheas of B. pseudohinzii-negative C57BL/6 J mice were explanted aseptically, surrounding tissue was removed and tracheas were cut into approximately 2 mm long pieces consisting of 2–3 cartilage rings. For electron microscopy, the rings were opened by cutting the trachealis muscle, washed in sterile PBS (Thermo Fisher Scientific) supplemented with penicillin (100 U/ml; PAA, Etobicoke, Canada) and streptomycin (0.1 mg/ml; PAA) and transferred into 6-well culture plates (Greiner bio–one, Kremsmünster, Austria). The rings were then submerged in phenol red–free MEM (Thermo Fisher Scientific) supplemented with penicillin, streptomycin and L-glutamine (2 mM) (Thermo Fischer Scientific) and cultured at 37 °C and 5% CO₂ for 24 h. After 24 h, viability of explants was validated by visualizing the beating of ciliated cells. Vital explants were then transferred into fresh plates and washed with subsequent addition of B. pseudohinzii cultures (strain 3227). Throughout, explants were kept in 1 ml of medium.
For measurements of PTS and CBF of in vitro-infected tracheal explants, each trachea was divided into 2 halves. They were transferred into a delta T-dish, washed with PBS, bacteria were added and the samples were incubated for 4 h at 37 °C. PTS and CBF were examined as described below. Throughout, explants were kept in 1 ml of medium.

BMMC and BMMØ were cultured as previously described in (5 (link)). Briefly, bone marrow was isolated from femurs of wild-type and transgenic mice. To initiate differentiation of BMMCs, bone marrow cells were cultured in IMDM supplemented with 10% heat-inactivated fetal calf serum (HI-FCS), 2 mM L-Glutamine (Sigma, G7513), 100 U/ml Penicillin + 100 µg/ml Streptomycin (Sigma, N109), 50 μM β-mercaptoethanol (Sigma M6250) and 2 ng/ml murine interleukin-3 (IL-3, Peprotech 213-13). Recombinant murine SCF (5 ng/ml, Peprotech 250-03) was added only during the first passage. c-kit⁺FcεRI⁺ BMMCs were used for experiments after 4 weeks of culture. Culture medium was supplemented with IL-3 (2 ng/ml) every two days.
To generate BMMØ, bone marrow cells were cultured in bacterial petri dishes (Greiner bio-one 633180) in RPMI supplemented with 10% HI-FCS, 2 mM L-Glutamine, 1% Penicillin/Streptomycin, 50 μM β-mercaptoethanol and 20% L929-conditioned medium. Non-adherent cells were collected after 5 days of culture to perform experiments.
For BMMC and BMMØ GPCR activation adenosine (Fluka Bio Chemika 01890), recombinant mouse C5a (R&D Systems #8085-C3-025) and platelet activating factor (PAF; Sigma, P7568) were used.

The DNAJB1-Luc-O23 cell line, a generous gift of Kampinga HH (Kampinga et al. 2009 (link); Wieten et al. 2010a (link)), was grown in DMEM supplemented with 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin, and 1 mg/mL hygromycin (Roche Diagnostics GmbH), in an 37 °C incubator with 5% CO₂.
After trypsinization, DNAJB-Luc-O23 cells were seeded into the wells of a white μClear 96-well plate (Greiner Bio-one, 1.5 × 10⁴ cells/well), and placed in DMEM medium with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. After 24 h, leucinostatin and sodium arsenite were added at specified concentrations (Fig. 7), and after o/n incubation, luciferase activity was measured with a Promega Steady-Glo Luciferase Assay System using a LB960 Microplate Luminometer (Berthold Technologies), according to the manufacturer’s instruction.

Peripheral blood (Institute for Transfusion Medicine, Jena University Hospital, Jena, Germany) was collected from healthy adult donors who had not taken any anti-inflammatory drugs during the last 10 days. The protocols for experiments with human monocytes were approved by the ethical committee of the Friedrich Schiller University Jena, Jena, Germany, on March 19, 2014; approval number: 4025-02/14. All methods were performed in accordance with the relevant guidelines and regulations. Leukocyte concentrates were centrifuged (4000 × g, 20 min, 20°C) and PBMCs were freshly isolated by dextran sedimentation and centrifugation on lymphocyte separation medium (Histopaque^®-1077, Sigma-Aldrich, Steinheim, Germany). Resulting PBMCs were seeded in RPMI 1640 (Sigma-Aldrich, Steinheim, Germany) supplemented with 10% heat inactivated FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin in cell culture flasks (Greiner Bio-one, Nuertingen, Germany) for 1–1.5 h at 37°C, 5% CO₂. Adherent monocytes were washed twice with PBS and finally harvested by cell scraping.