*4.1. Materials*

PNP (20 nm diameter, carboxylated and impregnated with near infrared (NIR) dye) was obtained from Thermo Fischer Scientific, Waltham, WA, USA). Ambient air pollution particles (diameter <0.20 μm (denoted as PM0.2)) were collected from air samples in downtown Los Angeles, CA, USA, per the protocol published elsewhere [43]. Transwell filters of 10.5 mm diameter (with 0.4 μm diameter pores), fetal bovine serum (FBS), and bovine serum albumin (BSA) were purchased from BD Biosciences (Franklin Lakes, NJ, USA). A 1:1 mixture of phenol red-free Dulbecco's modified Eagle's medium and Ham's F-12 medium (DME/F-12), nonessential amino acid solution (NEAA), *N*-(2-hydroxyethyl)piperazine-*N*- -(2-ethanesulfonic acid) hemisodium salt (HEPES), 2-(*N*-morpholino)ethanesulfonic acid sodium salt (MES), monodansylcadaverine (MDC), cytochalasin D (CCD), adenosine triphosphate (ATP), dimethylsulfoxide (DMSO), l-glutamine, 3-methyladenine (3-MA), bafilomycin, nocodazole, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), ciprofloxacin, trypsin-EDTA, Triton X-100, paraformaldehyde, chloroquine, and acridine orange (AO) were all obtained from Sigma-Aldrich (St. Louis, MO, USA). Primocin was purchased from InvivoGen (San Diego, CA, USA). 1,2-Bis(2-aminophenoxy)ethane-*N*,*N*,*N*- ,*N*- -tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) and Hoechst 33342 were obtained from Invitrogen (Carlsbad, CA, USA). Dylight (488 nm)-conjugated tomato lectin was obtained from Vector Laboratories (Burlingame, CA, USA). Some tomato lectin was labeled in-house using Dylight 405 NHS Ester labeling kit (Thermo Fischer Scientific). Fluo-8 AM was purchased from AAT Bioquest (Sunnyvale, CA, USA). BacMam LC3-GFP and Rab5a-GFP constructs, Lysotracker Green, and tetramethylrhodamine methyl ester (TMRM) were bought from Thermo Fischer Scientific. Microtubule-associated protein light chain 3B (LC3B) antibody (that recognizes both LC3B-I and -II) was purchased from Cell Signaling Technology (Danvers, MA, USA). Mitophagy detection kits (including the Mtphagy dye and lysosomal indicator (LI) dye) were obtained from Dojindo Molecular Technologies (Washington, DC, USA). A549 cells were purchased from American Type Culture Collection (Manassas, VA, USA).

#### *4.2. Cell Culture*

A549 cells were plated onto Transwell filters at 100,000 cells/0.865 cm2 and cultured in a defined medium with serum (MDS), composed of 10% FBS and serum-free defined medium (MDSF; DME/F-12 medium supplemented with 1 mM NEAA, 100 U/mL Primocin, 10 mM HEPES, 1.25 mg/mL BSA, and 2 mM l-glutamine). Cells were maintained at 37 ◦C in a humidified atmosphere of 95% air and 5% CO2 and fed every other day. Experiments were performed using A549 cells on culture days 2–3.

#### *4.3. Live Cell Imaging*

A549 cells cultured on Transwell filters were imaged by confocal microscopy as described elsewhere [26]. Briefly, A549 cells on Transwell filters were mounted in a temperature-controlled chamber (Vestavia Scientific, Vestavia Hills, AL, USA) and bathed with MDS on both sides. In xyz series, intracellular PNP fluorescence intensity was measured stack-by-stack and integrated over the entire volume of a single A549 cell. To demarcate intracellular space at the single cell level, cell plasma membranes were labeled using Dylight (488 or 405 nm)-conjugated tomato lectin. Confocal imaging was at 8 bits, 63× magnification, and 1024 × 1024 resolution with a SP8 confocal microscope system (Leica Microsystems GmbH, Wetzlar, Germany). Gallium nitride (405 nm), argon (488 nm), and helium-neon (633 nm) lasers were utilized for excitation. Image analysis was conducted using Image-J software (NIH, Bethesda, MD, USA) and Leica LAS 3D Process and Quantify Packages.

#### *4.4. Nanoparticle (NP) Exposure of A549 Cells*

PM0.2 at 1 μg/mL and near-infrared (NIR) dye-impregnated carboxylated PNP (20 nm diameter with excitation/emission (ex/em) wavelengths of 660/680 nm) at 80 μg/mL were used to apically expose A549 cells for 24 h unless noted otherwise.

#### *4.5. Intracellular PNP Content Assessed after 24 h of Apical PNP Exposure*

Incubation of PNP-exposed cells was performed in a humidified atmosphere of 5% CO2/95% air. At 24 h post exposure, cells were washed with fresh culture medium and intracellular PNP content was estimated using live cell imaging as above.

#### *4.6. E*ff*ect of Endocytosis Inhibitors on Intracellular PNP Content*

Intracellular PNP content was estimated by live cell imaging in the presence and absence of agents known to interfere with endocytosis pathways, including MDC (inhibiting clathrin-mediated endocytosis; 200 μM), CCD (inhibiting macropinocytosis; 10 μM), and nocodazole (inhibiting microtubule polymerization; 100 nM). A549 cells were exposed both apically and basolaterally to one of these inhibitors, beginning 30 min prior to and during apical PNP exposure of A549 cells, followed by intracellular PNP content assessment. Control represents PNP-exposed A549 cells studied in the absence of endocytosis inhibitors.

### *4.7. Egress of Intracellular PNP from A549 Cells*

A549 cells were apically exposed to 20 nm PNP at 80 μg/mL for 12 h, followed by washing thrice with fresh culture medium. A549 cells were then incubated on both sides with fresh MDS for up to 24 h. Intracellular PNP content of A549 cells at predesignated times was estimated to construct egress time courses.
