Approximately 5??105 cells were stained with Annexin V FITC (BD Biosciences) and propidium iodide (BD Biosciences) in 1 Annexin V binding buffer (BD Biosciences) following manufacturers protocols. F3 *test versus control: *contamination (Universal Mycoplasma Detection Kit, ATCC). ROS probe and requirements of oxidation products Hydroethidine (HE) was purchased from Invitrogen (Carlsbad, CA). A stock answer of HE (20?mM) was prepared in deoxygenated DMSO and stored at ?80?C until use. Ethidium cation (bromide salt) DPCPX was purchased from Sigma-Aldrich (St. Louis, MO). The hydroxylated oxidation product from HE (2-hydroxyethidium, 2-OH-E+) was prepared by reacting the probe with Fremys salt45,57. The dimeric product (E+CE+) was prepared by reacting the probe with extra potassium ferricyanide57. Synthesized requirements of all oxidation products of HE were purified by high-performance liquid chromatography (HPLC). HPLC analyses HPLC-based measurements of HE and its oxidation products were performed using an Agilent 1100 HPLC system (Santa Clara, CA) equipped with absorption and fluorescence detectors and a refrigerated autosampler (4?C). The samples DPCPX (50?L) were injected into a reverse phase column (Phenomenex, Kinetex C18, 100?mm??4.6?mm, 2.6?m) equilibrated with 20% acetonitrile (MeCN), 80% water containing 0.1% trifluoroacetic acid. The compounds were eluted by increasing the content of MeCN from 20 to 56% over 4.5?min at a flow rate of 1 1.5?mL/min. The detection parameters were as previously reported26,57. Monodansylcadaverine staining of autophagic vacuoles H2030 (7.5??103 cells/chamber) and H2030BrM3 (1.2??105 cells/chamber) were plated in eight-well glass chamber slides (Thermo Fisher Scientific) with RPMI complete medium, allowed to adhere for at least 24?h, and then treated with 2? M Mito-LND or DMSO ( 0.01%) dissolved in complete RPMI medium for 4?h. The growth medium was removed, and cells were stained with monodansylcadaverine (MDC; Sigma-Aldrich) for 30?min at 37?C to label acidic autophagic vacuoles. Cells were washed three times with phosphate-buffered saline (Thermo Fisher Scientific), and MDC fluorescence was visualized using the Cytation 5 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT) and the Eclipse Ts2 inverted fluorescent microscope (Nikon, Melville, NY) at 20 magnification with excitation/emission wavelengths of 460/535?nm. Lysate selections and Western blot analyses H2030, H2030BrM3, A549, NCI-H460, SAEC (Human Small Airway Epithelial Cells) and NHBE (Normal Human Bronchial Epithelial Cells) cells were seeded in T-25 flasks and adhered overnight prior to treatment with 2?M Mito-LND or DMSO dissolved in cell collection specific medium. To assess the impact of pharmacologically blocking autophagy, lung malignancy cells were pretreated with 50?M chloroquine diphosphate (Sigma-Aldrich) or vehicle (water) for 2?h prior to the addition of 1 1?M Mito-LND or vehicle (DMSO). To specifically DPCPX evaluate autophagy blockade via mitophagy inhibition, lung malignancy cells were pretreated with 5?M cyclosporin A (CsA; Sigma-Aldrich) or vehicle (DMSO) for 2?h prior to the addition of 2?M Mito-LND or vehicle (DMSO). Brightfield photomicrographs were obtained prior to lysate collection using an Olympus CK2 inverted microscope at 200 magnification. Cell lysates were prepared from cells harvested at 0, 6, 24, and 48?h post-treatment using lysis buffer (1% Triton X-100, 50?mM HEPES, pH 7.4, 150?mM NaCl, 1.5?mM MgCl2, 1?mM EGTA, 100?mM NaF, 10?mM sodium pyrophosphate, 1?mM sodium orthovanadate, 10% glycerol) with complete EDTA-free protease and PhosSTOP phosphatase inhibitors (Sigma-Aldrich). Protein was quantified using the DC protein assay (Bio-Rad, Hercules, CA). Approximately 20?g of protein was loaded in precast 4C20% Mini-Protean TGX gels (Bio-Rad), run for 1?h, transferred to a PVDF membrane with the Trans-Blot? Turbo? system (Bio-Rad) for 30?min, blocked for 1?h at room temperature, incubated overnight with primary antibodies, and incubated with the secondary antibody for.