*4.8. Cytosolic Ca2*<sup>+</sup> *Mobilization and PNP Exocytosis*

The effect of intracellular mobilization of Ca2<sup>+</sup> on PNP egress was assessed in washed A549 cells (after 12 h of apical PNP exposure as above) stimulated with 10 μM ATP added to both bathing fluids during the egress experiment for up to 24 h. Changes in cytosolic [Ca2<sup>+</sup>] were detected by Fluo-8 AM (1 μM, 10 min, 25 ◦C in the presence of 5 mM probenecid to prevent leakage of Fluo-8 from the cells) following a brief presence (for 2.5 min) of apically added 10 μM ATP.

#### *4.9. Nanoparticle Exposure-Induced Activation of Autophagy in A549 Cells*

A549 cells were apically exposed to NP (PNP or PM0.2) in the presence or absence of 40 μM chloroquine, which blocks autolysosome formation, that was added to both apical and basolateral bathing fluids 30 min prior to and during NP exposure. After 24 h NP exposure, A549 cells were washed with fresh culture medium thrice and fixed with 4% paraformaldehyde for 10 min at room temperature, followed by immunolabeling of autophagosomes using rabbit LC3B antibody (1:400 dilution, overnight, 4 ◦C) and Alexa 488-labeled goat anti-rabbit secondary antibody (1:400 dilution, 1 h, 25 ◦C). Antigen-antibody complexes were detected using confocal microscopy at ex/em of 488/500–550 nm.

#### *4.10. E*ff*ects of Inhibitors of Autophagosome or Autolysosome Formation on Intracellular PNP Content*

Intracellular PNP content was assessed in the presence of 3-MA (5 mM, an inhibitor of autophagosome formation) or bafilomycin (0.5 μM, an inhibitor of autolysosome formation). A549 cells were pre-incubated with one of these inhibitors in both apical and basolateral fluids 30 min prior to and during apical PNP exposure.

#### *4.11. Assessment of Lysosomal Dysfunction*

Lysosomal membrane permeability was assessed in A549 cells labeled with acridine orange (AO, 7 nM, 37 ◦C and 5% CO2) for 15 min [40]. Lysosomal dysfunction was detected by release of AO from lysosomes due to increased lysosomal membrane permeability (LMP), followed by accumulation of AO in cytosol and nucleus. In order to quantify LMP, an LMP index was estimated as AO green fluorescence intensity in cytoplasm and nucleus/total cellular AO green fluorescence intensity. Changes in AO fluorescence intensity in A549 cells were monitored using ex/em of 488/500–525 nm. A549 cells treated with ciprofloxacin (150 μM for 24 h) in both apical and basolateral bathing fluids was used as positive control.

#### *4.12. Assessment of Mitochondrial Function*

A549 cells were exposed to the mitochondrial membrane potential sensitive fluorescent dye TMRM (1 nM, ex/em: 561/570–630 nm) for 30 min in culture medium at 37 ◦C and 5% CO2 prior to live cell imaging. For the detection of mitophagy, A549 cells were apically exposed to Mtphagy dye (100 nM, ex/em: 561/650–800 nm) for 30 min in serum-free culture medium at 37 ◦C and 5% CO2. After labeling A549 cells with Mtphagy dye, cells were further apically exposed to NP (PNP or PM0.2), 1 μM FCCP (as a positive control) or DMSO (0.1% as a negative control) for 24 h in MDS. Thirty minutes prior to detection of mitophagy, bathing fluids of A549 cells were changed to MDSF containing LI lysosome marker dye (1 μM, ex/em: 488/490–550 nm). Live cell imaging was performed 30 min later for evidence of mitophagy (i.e., colocalization of the lysosomal marker dye with Mtphagy dye in lysosomes).

#### *4.13. Data Analysis*

Data are presented as mean ± standard deviation (*n* = total number of observations). Student's two-tailed *t*-tests were used for comparisons of two group means. One-way analysis of variance followed by post-hoc tests based on Tukey procedures was performed using Prism (version 6.07, GraphPad Software, La Jolla, CA, USA) to determine differences among means of ≥3 groups. *p* < 0.05 was considered statistically significant.

**Author Contributions:** Conceptualization, A.S., K.-J.K., and E.D.C.; methodology, A.S., K.-J.K., and E.D.C.; validation, A.S., K.-J.K., and E.D.C.; formal analysis, A.S., K.-J.K., and E.D.C.; investigation, A.S.; resources, K.-J.K., C.S., and E.D.C.; writing—original draft preparation, A.S. and K.-J.K.; writing—review and editing, A.S., K.-J.K., C.S., and E.D.C.; supervision, K.-J.K. and E.D.C.; project administration, K.-J.K. and E.D.C.; funding acquisition, E.D.C.

**Funding:** This work was supported in part by the Will Rogers Motion Picture Pioneers Foundation, Whittier Foundation, Hastings Foundation, and research grants (R01ES017034 and U01HL108364) from the National Institutes of Health.

**Acknowledgments:** Many thanks to Yang Chai for generous access to confocal microscopes. Edward D. Crandall is Hastings Professor of Medicine.

**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**

