*4.9. Isolation of Soluble Plasma Membrane Proteins*

The soluble fractions of PM proteins were obtained by ultracentrifugation, as previously described [55]. After normocapnia or hypercapnia treatment, cells were detached, homogenized in homogenization buffer (10 mM, 1 mM EDTA, 1 mM EGTA, 100 μg/mL N-tosyl-l-phenylalanine chloromethyl ketone, phosphatase, and protease inhibitors), and centrifuged at 500× *g* at 4 ◦C. Next, supernatants were collected and centrifuged at 100,000× *g* for 1 h at 4 ◦C. Afterward, the pellet, which contained the crude membrane fraction, was resuspended in homogenization buffer supplemented 1% Triton X-100 and centrifuged at 100,000× *g* for 30 min at 4 ◦C. After centrifugation, a supernatant containing a soluble membrane fraction was collected and used for the experiments.

### *4.10. Measurement of Na,K-ATPase Enzymatic Activity*

To measure ouabain-sensitive Na,K-ATPase activity, a high-sensitivity ATPase assay kit (Innova Biosciences, Cambridge, United Kingdom) was used, according to the instructions of the manufacturer, and was performed as described previously [55]. After treatment with normocapnia or hypercapnia, cells were detached, homogenized, and the PM fraction was isolated. To measure ouabain-sensitive ATPase activity, ouabain was applied to the soluble PM fractions and absorbance, which was measured at 600 nm by an Infinite M200 Pro reader (TECAN, Männedorf, Switzerland). Na,K-ATPase specific activity was calculated by subtraction of ouabain-sensitive from total ATPase activity.

#### *4.11. Detection of the Protein Oxidation*

Total protein oxidation was measured by using the OxyBlot protein oxidation detection kit (Merck Millipore, Darmstadt, Germany), according to instructions of the manufacturer. After normocapnia or hypercapnia treatment, cells were lysed, and an equal amount of proteins were derivatized by adding 1X 2,4-dinitrophenylhydrazine (DNPH) solution for 15 min. Afterward, a neutralization solution was added, samples were subjected to SDS-PAGE and transferred to nitrocellulose membranes. After incubation with antibodies from the kit, protein oxidation was determined by chemiluminescence and the density of detected protein oxidation was calculated by using the Image J software and normalized to the total protein amounts assessed by Coomassie staining.

#### *4.12. Measurement of Intracellular ATP Levels*

ATP levels after hypercapnia exposure were measured by an ATP bioluminescence assay kit HSII (Roche Diagnostics, Mannheim, Germany) following the instructions of the manufacturer. Cells were treated with normal or elevated CO2 levels, detached, and lysed in the lysis buffer provided with the kit. Next, samples were centrifuged at 10,000× *g* for 60 s and the supernatant was transferred to a fresh tube. Lastly, 50 μL of supernatants were transferred to 96-well plates. Additionally, 50 μL of the luciferase reagent was added to the samples and luminescence was measured immediately by Infinite M200 Pro reader. The obtained values were normalized to the protein amount as assessed by the Bradford assay.

#### *4.13. Measurement of Cellular Viability*

Cell viability was assessed by using the CASY cell counter and analyzer model TT (Roche Innovatis AG, Reutlingen, Germany) following the instructions of the manufacturer, as determined by an automatized measurement of plasma membrane integrity via an electric impulse. After exposure to normocapnic or hypercapnic conditions, cells were detached by incubation with 0.25% trypsin-EDTA, resuspended in culture medium, diluted in CASY tone solution, and the membrane integrity measurement was performed.

#### *4.14. Statistics*

Data are presented as mean ± SD unless otherwise indicated. Comparison of two groups was performed by paired (dependent) or unpaired (independent) two-tailed Student's t-test. Comparisons between more than two groups were performed by one-way or two-way analysis of variance (ANOVA) with a Dunnet test for multiple comparisons. For statistical analysis and data visualization, GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA) was used. A *p*-value of <0.05 was considered to be statistically significant.

**Author Contributions:** Conceptualization, V.K. and I.V. Data curation, V.K., R.E.M., S.H., W.S., O.V., and L.A.D. Formal analysis, V.K., O.V., and L.A.D. Funding acquisition, I.V. Investigation, V.K. and M.W. Methodology, V.K., M.W., O.V., and L.A.D. Project administration, M.W. and I.V. Resources, R.E.M., S.H., W.S., J.I.S., and I.V. Supervision, I.V. Validation, V.K., O.V., L.A.D., and J.I.S. Visualization, V.K. and I.V. Writing—original draft, V.K. and I.V. Writing—review & editing, V.K., R.E.M., S.H., W.S., O.V., L.A.D., J.I.S., and I.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** Grants from the Federal Ministry of Education and Research (German Center for Lung Research [DZL/ALI 3.1]), the Hessen State Ministry of Higher Education, Research and the Arts (Landes-Offensive zur Entwicklung Wissenschaftlichökonomischer Exzellenz [LOEWE]), the von Behring Röntgen Foundation (Project 66-LV07) and the German Research Foundation (DFG/KFO309, P5 and The Cardio-Pulmonary Institute (EXC 2026; Project ID: 390649896) (to I.V.) and a MD/PhD start-up grant (DFG/KFO309, MD/PhD) (to V.K.) as well as the National Institutes of Health (HL-147070) (to J.I.S.) supported this work.

**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.
