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Histology and Immunohistochemical Analyses
Lungs were perfused through the trachea with PBS or 4% paraformaldehyde (PFA), fixed overnight, transferred to 70% ethanol, embedded in paraffin, cut into 5-mm sections, and stained with hematoxylin/eosin. For immunohistochemical analyses, sections were incubated with ML 385 recognizing Ki-67 (RTU, RM-9106-R7, Thermo Scientific), NRF2 (ab137550, Abcam), SPC (AB3785, Millipore), HMGA2 (AP210AP, Biocheck), NKX2.1 (ab76013, Abcam) and processed with the Vectastain Elite ABC Kit (PK6101) and the DAB Peroxidase Substrate Kit (SK4100, Vector Laboratories). Histological slides were scanned with a Zeiss Axioplan 2 microscope (Carl Zeiss AG), and pathology and staining were quantified with TissueMorph software (Visiopharm Inte-grator System version 188.8.131.528). Tumor burden (tumor area/total lung area) on hematoxylin/eosin–stained slides was quantified with Visiopharm software (Visiopharm Integrator System version 184.108.40.2068).
Four-micrometer-thick sections of PFA inflation–fixed and paraffin-embedded lungs were deparaffinated, rehydrated, and loaded into a pressure cooker with DIVA Decloaker (DV2004; Biocare Medical) and cooled in Hot Rinse (HTR1001; Biocare Medical). Sections were then incubated in ice cold methanol for 1 min followed by normal goat serum (50197Z; ThermoFisher) and then by overnight in-cubation with an antibody against BACH1 (1:50; AF 5776; R&D Systems). Lungs were then incubated with donkey anti-goat IgG (H+L) Alexa-568 secondary antibody (1:250, A-11057, Life Technologies) for 1 hr. Sections were then mounted with Prolong Gold Antifade reagent with DAPI (P36935, Life Technologies) and imaged on a Zeiss LSM 700 confocal microscope with a 20xNA0.80 objective in 3x3 or 2x2 tiled fields. Images were acquired with ZEN software (2011, Carl Zeiss Microscopy, Jena, Germany).
Migration and Invasion Assays
Transwell migration and invasion assays were done with the xCELLigence system. For migration assays, cells were plated in CIM plates (05 665 817 001, ACEA Biosciences) (4 3 104 cells/well); for invasion assays, the CIM plates were precoated with a 1:40 dilu-tion of Matrigel. The plates were analyzed for 10 hr in the xCELLigence system, which monitors cells migrating or invading from serum-free conditions in an upper chamber through a microporous membrane to a lower chamber containing 10% serum, where cells are detected by biosensors. The electrode impedance is displayed as a migration or invasion index. Transwell migration assays were also done with inserts with a 6.5-mm, 8.0-mm-pore membrane. Cells (1 3 105/well) were suspended in serum-free medium in the upper chamber. The bottom chamber contained complete medium with 10% FBS. For invasion assays, the inserts were precoated with a 1:40 dilution of Matrigel. After 12 to 16 hr, cells in the upper chamber were removed with a humidified cotton swab, and migrating cells on the other side of the membrane were fixed with PFA, stained with crystal violet, and photographed under a bright-field microscope (10X). The area covered by cells was quantified with ImageJ on at least four random fields per well. Each experiment was done at least in duplicate.
Cell migration in real-time was also monitored with an IncuCyte ZOOM (Essen BioScience) (4 3 104 cells/well). This system mea-sures the closure of a scratched area in real time and automatically calculates the relative wound density within the initially vacant area at each time point. Cells were seeded in 96-well image-lock plates (4379, Essen BioScience). The next day, a scratch was made in each well with a WoundMaker (Essen Biosciences) which creates identical and reproducible scratches in all wells. After several washes to remove dead cells and debris, 200 mL of fresh medium containing the different drugs was added to each well. At least 3 experimental replicates were used per condition and cell line.
Cells were treated with NAC or Trolox (6-hydroxy-2,5,7,8-tetramethylchromane- 2-carboxylic acid; 238813, Sigma-Aldrich) for 7 days and seeded in white 96-well plates (5 3 103 cells/well). ROS were measured with the ROS-Glo-H2O2 assay (G8820, Promega). ROS were also measured in cells stably expressing ro-GFP2-ORP1 or roGFP2-Grx1 (Gutscher et al., 2009; Morgan et al., 2011). Cells were incubated for 1 week with antioxidants, and the day before analysis they were seeded in a 4-chamber, glass bottom, 35-mm CELLview dish (627871, Corning). Cells were washed and the medium was replaced with FluoroBrite medium (A18967-01, Life Technologies) before the assay. Fluorescence (excitation wavelengths, 405 and 488 nm; emission, 555–639 nm) in live cells main-tained at 37 C and 5% CO2 was recorded every 5 s with a Zeiss LSM 700 confocal microscope and a Plan-Achromat 40 3 /1.3 oil-immersion objective. Fluorescence intensity was determined with Zeiss Zen software.