*2.2. Influence of BLM Treatment on mRNA Expression of JAM-A and Localization in the Lung Tissue of P2X7*−/<sup>−</sup> *Mice in Comparison to the WT*

To investigate whether the BLM treatment in the lung tissue of P2X7−/<sup>−</sup> led to an altered expression of JAM-A in comparison to the WT animals, the PCLS of wildtype and P2X7−/<sup>−</sup> mice were prepared and treated with BLM for 24 h and 48 h (Figure 2). Using quantitative RT-PCR, we were able to demonstrate a marked increase in JAM-A expression in the PCLS of BLM-treated WT mice compared to BLM-treated PCLS of P2X7−/<sup>−</sup> mice (Figure 2, inset in A and E).

**Figure 2.** Paraffin sections from embedded PCLS after 300 mU/mL BLM exposure for 24 h (**A**,**B**,**E**,**F**) and 48 h (**C**,**D**,**G**,**H**). Immunoperoxidase demonstration of JAM-A in WT (**A**–**D**) and P2X7−/<sup>−</sup> (**E**–**H**) mice. Note the preferable immunostaining of AECII in untreated WT (arrows in **A**,**C**), a weak increase in P2X7−/<sup>−</sup> mice (**E**,**G**), and the strongest immunostaining of the AECs in the BLM-treated WT mice (**B**,**D**). Arrowheads depict the alveolar lining of JAM-A immunoreactivity. Bar = 100 μm. Inset over (**A**) and (**E**): Analysis of mRNA content in paraffin sections of PCLS from WT and P2X7−/<sup>−</sup> mice after 24 h. mRNA content of *JAM-A* was analyzed by quantitative real time RT-PCR using *Hmbs* and *Rpl32* as housekeeping genes. Charts are represented as mean ± SEM (WT normalized to 1; *n* = 3; *p*-value 0.4550).

Immunoperoxidase staining for JAM-A in the PCLS of WT lungs exhibited prominent staining at the AECI/II border and some additional cytoplasmic AECII staining, with increased immunoreactivity in the PCLS of P2X7−/−. The entire JAM-A immunoreactivity, as seen in frozen sections (compared to Figure 1), could not be reproduced in the paraffin sections, since fixation and paraffin embedding impaired the immunoreactivity and the structural conciseness of immunolocalization. The 24 h treatment with BLM led to a strongly enhanced immunoreactivity of the entire alveolar lining layer in the WT, but to a lesser extent in the P2X7−/<sup>−</sup> (Figure 2). This effect was stronger after 48 h of BLM treatment (Figure 2).

Some additional endothelial JAM-A immunostaining could not be excluded, since double staining with endothelial markers could not be performed in the present study due to the lack of suitable antibodies.

#### *2.3. The Inhibition of GSK-3*β *Leads to the Reduction of the Protein Content of JAM-A under BLM Treatment*

In the following experiment, different influences on JAM-A were evaluated. Based on the findings in the P2X7−/<sup>−</sup> mice where the inactivated form of GSK-3β, the GSK-3β(Ser9), was upregulated compared to WT [12], the GSK-3β(Ser9) protein level was investigated after BLM treatment in the alveolar epithelial E10 cells. In order to examine the role of GSK-3β in the expression of JAM-A, we used lithium chloride (LiCl), an inhibitor of GSK-3β, to treat the undamaged cells. Additionally, inactivation of GSK-3β under BLM treatment was performed to investigate the effects of inactivated GSK-3β on JAM-A under these conditions. After 24 h and 48 h of treatment with BLM, BLM + LiCl, or LiCl alone, the expression of the total GSK-3β was unchanged under all conditions when compared with the untreated E10 cells (Figure 3A).

**Figure 3.** Expression of total GSK-3β (**A**), GSK-3β(Ser9) (**B**), and JAM-A (**C**) were analyzed by Western blot after 24 h and 48 h treatment with 100 mU/mL BLM, 100 mU/mL BLM, and 10 mM LiCl or 10 mM LiCl alone. Equal protein amounts of cell lysates were used in SDS-PAGE and analyzed by Western blot. α-Tub served as the loading control. Untreated cells were used as the control and normalized to 100%. Representative blots from three independent experiments are shown. Charts presented as mean ± SEM (*n* = 3) of GSK-3β /α-Tub, GSK-3β(Ser9)/α-Tub, and JAM-A/α-Tub. *P*-values: 24 h GSK-3β 0.9447; 48 h GSK-3β 0.0625; 24 h GSK-3β(Ser9) 0.0046; 48 h GSK-3β(Ser9) 0.0066; 24 h JAM-A 0.186; and 48 h JAM-A 0.6123. \* *p* < 0.05, \*\* *p* < 0.01.

After 24 h of BLM treatment, no increase in GSK-3β(Ser9) was seen (Figure 3B). The addition of LiCl to BLM or LiCl alone increased the expression of GSK-3β(Ser9) only slightly (Figure 3B), but significantly in comparison to the BLM-treated cells.

As shown in Figure 3C, 24 h of BLM treatment of the E10 cells induced an increase of 132.3% in JAM-A. Inhibition of GSK-3β with LiCl in combination with BLM reduced the increase of JAM-A, indicating that the inactivation of the GSK-3β under BLM treatment had consequences for the expression of JAM-A (Figure 3C). Treatment of E10 cells with LiCl alone also downregulated the protein content of JAM-A to 75.6% compared to the untreated control cells.

After 48 h of BLM treatment, no increase in GSK-3β(Ser9) was also seen (Figure 3B). The addition of LiCl after 48 h of BLM treatment resulted in a strong increase in the protein content of the inactive form GSK-3β(Ser9) to 178.5% compared to the BLM-treated cells (Figure 3B). Likewise, treatment with LiCl alone led to a dramatic increase in GSK-3β(Ser9).

After 48 h of BLM treatment, the early increase in JAM-A returned to the level seen in the control cells (Figure 3C) The strong upregulation of GSK-3β(Ser9) under BLM treatment or by sole LiCl treatment in the control cells did not lead to any changes in the JAM-A protein.

In summary, early BLM treatment (24 h) in alveolar epithelial cells induced an increase in JAM-A. The BLM treatment did not inactivate GSK-3β within a period of 48 h. The early rise of JAM-A after BLM exposure could be reduced to the protein level of the control cells by inactivation of GSK-3β. This means that the upregulation of the inactive form of GSK-3β prevents the rise of JAM-A under BLM treatment.

### *2.4. The P2X7R Indirectly Regulates JAM-A Protein Content by the Modulation of GSK-3*β*(Ser9)*

The aim of the following experiment in the E10 cells was to investigate how the inhibition of P2X7R under BLM treatment (24 h) affected the inactive form of GSK-3β and whether subsequent changes in JAM-A occurred. The increase in the protein content of P2X7R after BLM treatment in the E10 cells has already been shown in one of our previous studies [17]. Additionally, the increase in JAM-A after BLM treatment was confirmed (Figure 3) and there was no inactivation of GSK-3β at this time when compared to the untreated control cells (Figure 3).

To test the hypothesis that P2X7R could indirectly modulate JAM-A following BLM treatment by regulating the inactive form of GSK-3β, we studied the GSK-3β(Ser9) and JAM-A protein contents after inhibition of P2X7R by oxATP (Figure 4).

The inhibition of P2X7R under BLM treatment led to a strong decrease in the GSK-3β(Ser9) protein content in the alveolar epithelial cells in comparison to the untreated or BLM-treated cells. The significant reduction in GSK-3β(Ser9) protein content compared to BLM-treated cells resulted in a re-upregulation of JAM-A. Treatment with oxATP alone led to significant downregulation of GSK-3β(Ser9) when compared to the untreated cells. The inhibition of P2X7R under these conditions also produced an increase in JAM-A when compared to the untreated cells.
