*4.3. Cell Line and Cell Culture*

The mouse lung cell line E10 [30] was acquired by M. Williams (Pulmonary Center, Boston University School of Medicine, Boston, MA, USA). Cells were cultured in DMEM/Ham's F12 medium (1:1) purchased by Gibco (ThermoFisher Scientific, Waltham, MA, USA), supplemented with 5% (*v*/*v*) fetal bovine serum (Biochrom AG Seromed, Berlin, Germany) and 2.5 mM L-glutamine (Merck KGaA, Darmstadt, Germany). E10 cells were seeded at a density of 1.5 <sup>×</sup> 104 to 3 <sup>×</sup> 10<sup>4</sup> cells/mL and passaged three times a week up to 25 passages and were grown at 37 ◦C in a 5% CO2 atmosphere up to a confluence of 60–80%.

For treatment of the cells, the medium was supplemented with 100 mU/mL BLM (Bleocell, STADA Arzneimittel AG, Bad Vilbel, Germany), 150 μM oxATP, 150 μM BzATP, and 10 mM LiCl (Sigma-Aldrich, Munich, Germany). OxATP and LiCl were added to the cells 1 h before BLM treatment.

### *4.4. Precision-Cut Lung Slices (PCLS) and Tissue Culture*

To attain PCLS, we followed the procedure published by [31] with several modifications such as the process of obtaining, cutting, handling, and incubating the lung slices described in detail by [8]. After 24 h of incubation, the lung slices were homogenized for real-time reverse transcription PCR, Western blot analyses, or embedded in paraffin for immunohistochemistry.

#### *4.5. RNA Isolation and Real-Time Reverse Transcription PCR (Real-Time RT PCR)*

The isolation of total RNA was realized as previously described with some modification [8]. Primers applied for real-time RT PCR were: JAM-A (5´-TCCTGGGCTCTTTGGTACAAGG-3, 5´-TCCTGGGCTCTTTGGTACAAGG-3), mHmbs (5´-GCTTCGCTGCATTGCTGAAA-3´, 5´-CCAGTCAGGTACAGTTGCCC-3´), mRpl32 (5´-GCACCAGTCAGACCGATATGTG-3´, 5´-CTTCTCCGCACCCTGTTGTC-3´).

The identity of the PCR products was proven by melting point analysis. Using the ΔΔCT method at CFX Manager (Bio-Rad), the relative quantification of gene expression was performed with housekeeping genes *Hmbs* (hydroxymethylbilane synthase) and Rpl32 (ribosomal protein L32).

#### *4.6. Western Blot Analysis*

Total protein concentrations of the lysates of lung tissue and cells were determined by using the BCA Protein Assay Kit (ThermoFisher Scientific) according to the manufacture's guidelines. Murine tissue protein extraction was performed as described previously in [12] by using the Precellys 24 Homogenizer (PEQLAB, Erlangen, Germany) and lysis buffer including 0.02 M Tris, pH 8.5; 0.125 M NaCl; and 1% (*v*/*v*) Triton X-100 with protease inhibitor Complete Tablets, EDTA-free (Roche, Basel, Switzerland).

Cell protein extraction was performed by applying ice cold PBS buffer without Mg2<sup>+</sup> and Ca2<sup>+</sup> (Biochrom AG, Berlin, Germany) supplemented with protease inhibitor Complete Tablets, EDTA-free (Roche) into T75 flasks (Greiner Bio-one GmbH, Frickenhausen, Germany). The cells were scratched from the surface, collected, and pelleted at 500× *g* for 5 min. After adding lysis buffer including 0.02 M Tris, pH 7.5; 0.14 M NaCl; 1 mM EDTA, pH 8.0; and 1% (*v*/*v*) Triton X-100 with protease inhibitor Complete Tablets, EDTA-free (Roche), the lysate was incubated 45 min at 4 ◦C with slight motion and centrifuged at 10.000× *g* for 20 min at 4 ◦C to collect the supernatant containing the protein.

For SDS-PAGE, the samples were diluted to a homogeneous total protein concentration, transferred into 6× SDS sample buffer (300 mM Tris, pH 6.8; 100 mM Dithiothreitol; 0.1% (*w*/*v*); 30% (*w*/*v*) glycerol; 10% (*w*/*v*) SDS), boiled at 95 ◦C for 5 min and 10–50 μg of total protein per sample were loaded on a 12% SDS-polyacrylamide gel. Western blot analysis was performed as described [32] with minor modifications. The PVDF Immobilon-P Membrane (Merck Millipore, Billerica, USA) was blocked for 1 h in TBS-T buffer (17 mM Tris, pH 7.4; 2.7 mM KCl; 137 mM NaCl; 0.2% (*v*/*v*) Tween 20) including 5% (*w*/*v*) dried non-fat powdered milk (Carl Roth GmbH, Karlsruhe, Germany) and incubated at 4 ◦C overnight with the following primary antibodies: polyclonal rabbit anti-human JAM-A (A302-891A), dilution 1:1000 (Bethyl Laboratories Inc.); polyclonal rabbit anti mouse P2X7R (APR-004; Alomone Labs Inc.), dilution 1:500; monoclonal mouse anti-human GSK-3β (610202; BD Biosciences), dilution 1:1000; monoclonal rabbit anti-human Phospho-GSK-3β (Ser9) (#9323; Cell Signaling Technology Inc.), dilution 1:1000; and monoclonal mouse anti -tubulin (sc-8035; Santa Cruz Biotechnology Inc.), dilution 1:1000.

The following secondary antibodies were used at room temperature for 1 h: donkey anti-rabbit IgG, HRP-linked F(ab)2 fragment (GE Healthcare, Chalfont St Giles, Buckinghamshire, UK), and horse anti-mouse IgG, HRP-linked antibody (Cell Signaling, Danvers, MA, USA). The chemiluminescent signal was detected by using Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore) by following the manufacturer's guidelines and Image Reader LAS-3000 (Fujifilm, Tokyo, Japan). Quantification was done with ImageJ 1.51 u free software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) and each line was normalized to corresponding α-tubulin (α-Tub).

#### *4.7. Immunohistochemistry*

Fixation of 5 μm thin lung tissue in 4% (*v*/*v*) buffered formalin, embedding in paraffin, sectioning, and immunostaining were performed as described previously in [8,17]. The primary antibody against JAM-A was detected with a biotinylated secondary antibody, followed by incubation with the streptavidin/biotin-peroxidase complex (Vectastain Elite Kit, Serva, Heidelberg, Germany). As a negative control, the primary antibody was replaced with PBS or a non-immune serum.

### *4.8. Immunofluorescence*

Immunofluorescence and double immunofluorescence staining on frozen cryostat sections of mouse lung tissue were performed, as previously described in Reference [33]. The following primary antibodies were used: polyclonal rabbit anti JAM-A and monoclonal mouse anti T1α clone E11, dilution 1:200, which was a kind gift from Dr. A. Wetterwald, Bern (Switzerland). As secondary antibodies, we used goat anti-rabbit IgG conjugated to fluorescein isothiocyanate (FITC, Dianova, Hamburg, Germany; dilution 1:100 (*v*/*v*)) and donkey anti-mouse IgG, Texas Red labelled (Dianova, Hamburg, Germany; dilution 1:100 (*v*/*v*)). Negative controls included the omission of the primary antibody. Acetone-methanol fixed monolayers of E10 cells were similarly incubated with the same dilution of the JAM-A antibody.

#### *4.9. Statistical Analysis*

One-way analysis of variance (ANOVA) was used to determine the results of Western blot analysis and real-time RT PCR with three or more groups, followed by the post hoc Bonferroni test in case significance was achieved. Statistical comparison of two groups was performed as a two tailed Student´s *t* test. Statistical analysis was done with GraphPad Prism 5.03 software (GraphPad Software, San Diego, CA, USA). We determined significance at \* *p* = 0,05 and high significance at \*\* *p* = 0.01. A minimum of three independent experiments for each technique was performed.

**Author Contributions:** Conceptualization, K.B. and M.K.; Investigation, K-P.W. and A.S.; Data curation, K-P.W. and M.K.; Writing, K-P.W., M.K., and K.B.; Writing–review & editing, K.B.

**Funding:** This work was supported by the Deutsche Forschungsgemeinschaft (DFG), BA 3899/4-1.

**Acknowledgments:** The authors thank A. Neisser, S. Bramke, and D. Streichert for their expert technical assistance and Heinz Fehrenbach (Borstel, Germany) for his critical reading of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **Abbreviations**

BLM Bleomycin GSK-3β Glycogen synthase kinase-3 beta JAM-A Junctional adhesion molecule-A LiCl Lithium chloride PCLS Precision-cut lung slices PKC-β1 Protein kinase C-beta1 P2X7R P2X7 receptor WT Wildtype
