Human NSCLC cell lines A549, H1975, H838, H1299, H23, and H460 were purchased from the American Type Culture Collection (ATCC). Mouse lung tumor cells lines were isolated from Kras2 LSL/+ mice with the tumor dissociation kit for mouse (130-096-730, MACS Miltenyi Biotec) and the gentleMACS Octo Dissociator (130-096-427, MACS Miltenyi Biotec) 58 weeks after administration of low-dose Cre-adenovirus. Human and mouse lung cancer cells were cultured in DMEM low-glucose GlutaMax medium (21885-108, Life Technologies) supplemented with 1% NEAA (11140-035, Life Technologies), 1% penicillin-streptomycin (15140122, Thermo Fisher Scientific), and 10% fetal bovine serum (FBS, 10270106, Thermo Fisher Scientific). Virus-packaging cells (293FT and GP2-293) were cultured in DMEM high-glucose GlutaMAX (31966047, Thermo Fisher Scientific), supplemented with 1% penicillin-streptomycin and 10% FBS. All cell lines were negative for mycoplasma. No cell lines used in this study were found in the database of commonly misidentified cell lines that is maintained by ICLAC and NCBI Biosample.

Kras2Trp53mice (designated KP) () were maintained on a mixed C57BL/6-129/Sv genetic background; littermates were used as controls. Mice 6–8 weeks old were randomly selected to inhale Cre-adenovirus (University of Iowa, Iowa City, IA) intranasally. pSECC virus was delivered intratracheally. N-acetylcysteine (NAC, A7250, ≥ 99% purity, Sigma) was administered in the drinking water (1 g/L). Vitamin E (DL-α-tocopheryl acetate) was administered in the chow (Lantmännen) at a dose of 0.5 g/kg chow (61.5 mg/kg body weight), calculated from observed daily food intake. For allograft experiments, mouse lung tumor cells were transplanted intravenously into syngeneic mice (1 × 10cells) or NOD-SCID-gamma mice (NSG; NOD.Cg-PrkdcIl2rg/SzJ) (0.5 × 10cells); mice were killed and lungs harvested 3 weeks later. Cells (2.5 × 10were transplanted subcutaneously into NSG mice. For transplantation of human NSCLC cells, 5 × 10cells were injected intravenously, and lungs were harvested 10 weeks later. For 3-bromopyruvate (3-BP) and AZD3965 experiments, cells were pre-treated in vitro with the inhibitors for 3 days before injection into mice. After cell injection, 3-BP (10 mg/kg) or vehicle (PBS) was administered daily intraperitoneally for 4 days. Alternatively, mice were treated with AZD3965 (100 mg/kg) or vehicle (PBS, 0.1% Tween 80, hydroxypropyl methylcellulose 0.5%) by oral gavage for 4 days. Animal experiments were approved by the Research Animal committees in Gothenburg and Linköping, Sweden.

Methods Details

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-μm sections, and stained with hematoxylin/eosin. For immunohistochemical analyses, sections were incubated with antibodies 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 Integrator System version 5.0.2.1158). Tumor burden (tumor area/total lung area) on hematoxylin/eosin–stained slides was quantified with Visiopharm software (Visiopharm Integrator System version 5.0.2.1158).

Immunohistofluorescence 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 incubation 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 × 104 cells/well); for invasion assays, the CIM plates were precoated with a 1:40 dilution 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-μm-pore membrane. Cells (1 × 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 brightfield 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 × 104 cells/well). This system measures 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 μL of fresh medium containing the different drugs was added to each well. At least 3 experimental replicates were used per condition and cell line.

ROS Measurements 3 cells/well). ROS were measured with the ROS-Glo-H 2 O 2 assay (G8820, Promega). ROS were also measured in cells stably expressing ro-GFP2-ORP1 or roGFP2-Grx1 ( Gutscher et al., 2009 Gutscher M.

Sobotta M.C.

Wabnitz G.H.

Ballikaya S.

Meyer A.J.

Samstag Y.

Dick T.P. Proximity-based protein thiol oxidation by H2O2-scavenging peroxidases. Morgan et al., 2011 Morgan B.

Sobotta M.C.

Dick T.P. Measuring E(GSH) and H2O2 with roGFP2-based redox probes. 2 was recorded every 5 s with a Zeiss LSM 700 confocal microscope and a Plan-Achromat 40 × /1.3 oil-immersion objective. Fluorescence intensity was determined with Zeiss Zen software. 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 × 10cells/well). ROS were measured with the ROS-Glo-Hassay (G8820, Promega). ROS were also measured in cells stably expressing ro-GFP2-ORP1 or roGFP2-Grx1 (). 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 maintained at 37°C and 5% COwas recorded every 5 s with a Zeiss LSM 700 confocal microscope and a Plan-Achromat 40 × /1.3 oil-immersion objective. Fluorescence intensity was determined with Zeiss Zen software.

GSH, NADH and NADPH Measurements Cells were incubated with NAC or Trolox for 7 days and seeded in white 96-well plates (5–10 × 103 cells/well). The next day, total glutathione and NADH and NADPH levels were measured with GSH-Glo (V6911) and NAD(P)H-Glo (G9061, G9062 Promega) assays. The ratio of GSH/GSSG was determined with the GSH/GSSG-Glo assay (V6611, Promega).

Free Heme Measurements Espinas et al., 2012 Espinas N.A.

Kobayashi K.

Takahashi S.

Mochizuki N.

Masuda T. Evaluation of unbound free heme in plant cells by differential acetone extraction. Free heme was extracted with neutral acetone as described (). Heme from neutral acetone extracts was quantified with the Heme assay kit (MAK316-1KT, Sigma-Aldrich), and measurements were normalized to protein concentration. To induce release of free heme by ROS, cells were treated with 200 μM hydrogen peroxide overnight. Hanna et al., 2016 Hanna D.A.

Harvey R.M.

Martinez-Guzman O.

Yuan X.

Chandrasekharan B.

Raju G.

Outten F.W.

Hamza I.

Reddi A.R. Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors. 4 cells per well of a 96-well plate were reverse transfected with X-tremeGENE HP DNA Transfection Reagent (6366244001, Sigma-Aldrich) at a 1:3 DNA:Transfection Reagent ratio and seeded together with medium supplemented with antioxidant, vehicle, or pro-oxidant (diamide, 5 μM, D3648-1G, Sigma-Aldrich). Cells were incubated at 37°C for 48 hr before eGFP and mKATE2 imaging (HCS Operetta, Perkin-Elmer). The values presented correspond to 3 transfections per treatment. Each data point is the average of the fluorescence intensity ratio of eGFP to mKATE2 of 23 fields of view. For labile heme measurements with a heme biosensor, A549 cells were treated for 18 days with NAC, Trolox, or vehicle (water and DMSO, respectively) and transfected with the genetically encoded heme sensor pCDNA-HS1 (). Briefly, 5.6 × 10cells per well of a 96-well plate were reverse transfected with X-tremeGENE HP DNA Transfection Reagent (6366244001, Sigma-Aldrich) at a 1:3 DNA:Transfection Reagent ratio and seeded together with medium supplemented with antioxidant, vehicle, or pro-oxidant (diamide, 5 μM, D3648-1G, Sigma-Aldrich). Cells were incubated at 37°C for 48 hr before eGFP and mKATE2 imaging (HCS Operetta, Perkin-Elmer). The values presented correspond to 3 transfections per treatment. Each data point is the average of the fluorescence intensity ratio of eGFP to mKATE2 of 23 fields of view.

Lentiviral Production and Transduction Joung et al., 2017 Joung J.

Konermann S.

Gootenberg J.S.

Abudayyeh O.O.

Platt R.J.

Brigham M.D.

Sanjana N.E.

Zhang F. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Gitenay et al., 2014 Gitenay D.

Wiel C.

Lallet-Daher H.

Vindrieux D.

Aubert S.

Payen L.

Simonnet H.

Bernard D. Glucose metabolism and hexosamine pathway regulate oncogene-induced senescence. ; pLPCx-Grx1-roGFP2 and pLPCx-roGFP2-Orp1 were from T. Dick ( Gutscher et al., 2009 Gutscher M.

Sobotta M.C.

Wabnitz G.H.

Ballikaya S.

Meyer A.J.

Samstag Y.

Dick T.P. Proximity-based protein thiol oxidation by H2O2-scavenging peroxidases. Morgan et al., 2011 Morgan B.

Sobotta M.C.

Dick T.P. Measuring E(GSH) and H2O2 with roGFP2-based redox probes. Lentiviruses were produced by transfecting 293FT packaging cells (R70007, Life Technologies) with lentiviral backbone constructs (6 μg), packaging plasmid psPAX2 (Addgene plasmid #12260, 3 μg), and envelope plasmid pMD2.G (Addgene plasmid #12259, 1.5 μg) using the Xfect transfection reagent (631318, Clonetech). Retroviruses were produced by transfecting GP2-293 packaging cells (6314588, Clonetech) with retroviral backbone (5 μg) and envelope plasmid (1 μg). Lentiviral and retroviral supernatants were collected 2 days after transfection. Target cells were transduced once with lentiviruses supplemented with 8 μg/ml polybrene (107689-10G, hexadimethrine bromide; Sigma-Aldrich) and selected with blasticidin (1–5 μg/mL, A1113903), hygromycin B (100 μg/mL, 10687010), or puromycin (1 μg/mL, A1113803, ThermoFisher Scientific). Lentiviral backbones were pLentiCRISPRv2blast (#98293, Addgene) expressing single-guide (sg) RNA targeting genes of interest, or pLentiMPHv2 (#89308, Addgene), pLentiSAMv2 (#75112, Addgene) as described (). Retroviral plasmids were pWZL-HK2-Flag (#20501) and pLPC-G6PC3 (pLPCx-Grx1-roGFP2 and pLPCx-roGFP2-Orp1 were from T. Dick (). Sánchez-Rivera et al., 2014 Sánchez-Rivera F.J.

Papagiannakopoulos T.

Romero R.

Tammela T.

Bauer M.R.

Bhutkar A.

Joshi N.S.

Subbaraj L.

Bronson R.T.

Xue W.

Jacks T. Rapid modelling of cooperating genetic events in cancer through somatic genome editing. For sgRNA cloning, the lentiCRISPRv2 vector was digested with BsmBI and ligated with BsmBI-compatible pre-annealed oligonucleotides. The following sequences were used for CRISPR-knockout strategies: sgdTomato GGCCACGAGTTCGAGATCGA; mouse sgBach1_m1 GTACTTCCACTCGAGAA-TCGT; sgBach1_m2 GTCTGGCCTACGATTCTCGAG; human sgBACH1_h1 CCTGGCCTACGATTCTTGAG, sgBACH1_h2 CCACTCAAGAATCGTAGGCC; and sgBACH1_h3 TACTCAGCCTTAAT-GACCAG. For the transcriptional activation strategy with dCAS9-SAM, the following sequences were used: sgdTomtato, GGCCACGAGTTCGAGATCGA; mouse sgBach1_m2, GGCCCGGGGGCGGAAC-CGG; and human sgBACH1_h2, GACACATCAGCACCGCCCTCG. Expression of target proteins in CRISPR-knockout and CRISPR-SAM experiments was evaluated by western blotting 3–5 days after selection. The pSECC lentiviral vector and cloning strategy are described elsewhere (

Western Blotting Cells were lysed in buffer containing 9 M urea and Halt protease inhibitor cocktail (78430, Life Technologies) and Halt phosphatase inhibitor (78428, Life Technologies). Alternatively, cells were lysed in Laemmli buffer supplemented with β-mercaptoethanol. Cytosolic and nuclear extracts were prepared with NE-PER nuclear and cytoplasmic extraction reagents (78835, Life Technologies). Protein concentration of lysates was determined with the Pierce BCA Protein Assay Kit (23225, Life Technologies). After denaturation, equal amounts of proteins were resolved on 4%–20% or 12% Mini-PROTEAN TGX Stain-Free gels (BioRad), and electro-transferred onto nitrocellulose membranes. The membranes were blocked with TBST containing 5% milk and incubated with antibodies against BACH1 (sc-271211, Santa Cruz Biotechnology, 1:1000), HO-1 (MA1-112, Life Technologies, 1:1000), HK2 (PA5-29326, Life Technologies, 1:2000), β-actin (A228, Sigma-Aldrich), GAPDH (G9295, Sigma-Aldrich, 1:1000), histone 3 (ab1791, Abcam, 1:5000), NQO1 (HPA007308, Sigma-Aldrich, 1:200), KEAP1 (#8047S, Cell Signaling, 1:1000), and NRF2 (#12721, Cell Signaling, 1:300). Secondary antibodies were from Jackson Immunoresearch laboratories. Clarity Western ECL substrate (1705061, Bio-Rad) was used for detection with the ChemiDoc Touch Imaging system (1708370, Bio-Rad).

Cell Viability Assay Cell viability was estimated with Presto Blue Cell Viability Reagent (A13262, Life Technologies). Cells (5000/well) were seeded in 96-well plates in triplicate. After 24–72 hr, PrestoBlue reagent (10 μl) was added to wells with cells or medium (blank), and absorption at 570 and 600 nm was measured with a Synergy plate reader (BioTek). To assess the effect of KI-696 on cell viability, 2.2 × 104 cells per well of a 24-well plate were treated with KI-696 (5 μM) or vehicle (DMSO). Cells were incubated at 37°C for 48 hr before assay of cell viability (Vi-Cell XR, Beckman-Coulter).

Extracellular Flux Measurements Metabolic analyses were done with the Seahorse XFe96 Analyzer (Agilent), which measures the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of live cells. Glycolytic rates were measured with the Seahorse XF glycolytic rate assay (S7805A, Seahorse Agilent), ATP production rates were measured with the Seahorse XF ATP rate (103592-100, Seahorse Agilent): cells were seeded in Seahorse XF96 microplates (15,000 cells/well) (101085-004, Agilent) and cultured overnight at 37°C in a CO 2 incubator. On the day of the assay, the medium was replaced with freshly prepared phenol red–free base medium supplemented with HEPES, glucose, pyruvate, and glutamine and adjusted to pH 7.4. Cells were incubated for 45 min at 37°C in a non-CO 2 incubator before the assay, and the medium was changed immediately before the assay. Before assays, the Seahorse Analyzer pre-warmed to 37°C. Data were normalized to cell numbers obtained from additional wells using Presto Blue Cell Viability. Glycolytic and ATP production rates were calculated with the Macro Excel file provided by the manufacturer.

Total ATP, Glucose Uptake and Lactate Secretion Glucose uptake and total ATP were measured on cells seeded in white 96-well plates (5000 cells/well) (Glucose uptake-Glo J1342, Cell-Titer-Glo, G7571, Promega). Extracellular lactate secretion was measured by collecting medium from 5000 cells cultured for 24 hr in 96-well plates. Harvested medium was diluted in PBS (1:50), and lactate content was determined with the Lactate-Glo assay (J5021, Promega) using 10 μL of diluted supernatant. Cells remaining in the 96-well plates were used to normalize the data.

RNA-Sequencing and Bioinformatics Dobin et al., 2013 Dobin A.

Davis C.A.

Schlesinger F.

Drenkow J.

Zaleski C.

Jha S.

Batut P.

Chaisson M.

Gingeras T.R. STAR: ultrafast universal RNA-seq aligner. Anders et al., 2015 Anders S.

Pyl P.T.

Huber W. HTSeq--a Python framework to work with high-throughput sequencing data. Love et al., 2014 Love M.I.

Huber W.

Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Yu et al., 2012 Yu G.

Wang L.-G.

Han Y.

He Q.-Y. clusterProfiler: an R package for comparing biological themes among gene clusters. Total RNAs were extracted with the RNeasy Plus Mini kit (74136, QIAGEN). Sequencing was done with an Illumina HiSeq PE150 instrument and Promega Relia-Prep. Paired end reads were obtained, and their quality score was determined with FASTQC. Sequencing reads were mapped to the GRCm38/mm10 mouse genome assembly with STAR Aligner (version 2.5.0a) (), and genes were quantified with the HTSeq-count tool (version 0.6.1) (). Tests for differential expression of genes identified in RNA-seq analyses were done with DESeq2 (). Differentially expressed genes with a false-discovery rate < 0.05 were considered statistically significant. R Bioconductor package clusterProfiler was used for enrichment analyses (). Putative promoter regions 1000 bp upstream of each differentially regulated gene (n = 1025) were successfully extracted for 961 genes. Both Mus musculus BACH1 and NRF2 binding motifs were downloaded from JASPAR database of transcription factor binding profiles by using R package MotifDb. Sequences 1000 bp upstream of genes were matched by the position-weighted matrix method using a multinomial model with a Dirichlet conjugate before calculation of estimated probability of base b at the position. The threshold of matching was set to 75%. Details about the method have been described (Bioconductor Maintainer at https://bioconductor.org/packages/devel/workflows/vignettes/generegulation/inst/doc/generegulation.html ). The SEEK database ( http://seek.princeton.edu ) was interrogated to retrieve genes co-expressed with BACH1 in human lung cancer datasets (173 datasets). TGCA data were downloaded from http://www.cbioportal.org/ . STRING analysis was done at https://version-10-5.string-db.org/ (version 10.5) using default settings, medium confidence (0.400), and four clusters.

Real-Time Quantitative PCR RNA was isolated with the RNeasy Plus Mini kit (74136, QIAGEN), and cDNA was synthesized with the iScript cDNA synthesis kit (170-889, Bio-Rad). Gene expression was analyzed by TaqMan reverse transcription quantitative polymerase chain reaction (qPCR) on a CFX384 Real-Time System (Biorad) using mouse and human probe sets for Bach1 Mm01344527_m1, Hk1 Mm00439344_m1, Hk2 Mm00443385_m1, Mmp1a Mm00473485_m1, Dusp1 Mm00457274_g1, Gapdh Mm03302249_g1, Hmox1 Mm00516005_m1, Gclm Mm01324400_m1, Sqstm1 Mm00448091_m1, Nqo1 Mm01253561_m1, Slc16a1 Mm01306379_m1, Pkp3 Mm00451123_m1, Rplp0 Mm00725448_s1. After screening a panel of murine reference genes (TATAA Biocenter) and analyzing variability with GenEx, we selected ribosomal protein lateral stalk subunit P0 (Rplp0) as a reference gene. Reactions were done in triplicate.

Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) Lung tumor mTC and mTN cells (n = 3 biological replicates per condition) were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was isolated by adding lysis buffer, and the cells were disrupted with a Dounce homogenizer. Lysates were sonicated, and the DNA was sheared to an average length of 300–500 bp. Genomic DNA (input) was prepared by incubating chromatin aliquots with RNase and proteinase K, heated for de-crosslinking, and precipitated with ethanol. Pellets were resuspended, and the resulting DNA was quantified with a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (30 μg) was pre-cleared with protein G agarose beads (Life Technologies). Genomic DNA regions of interest were isolated with 4 μg of antibody recognizing BACH1 (AF577, R&D Systems). Complexes were washed, eluted from the beads with sodium dodecyl sulfate buffer, and incubated with RNase and proteinase K. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Illumina sequencing libraries were prepared from the ChIP and input DNAs by the standard consecutive enzymatic steps of end-polishing, dA addition, and adaptor ligation. After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced with an Illumina NextSeq 500 (75-nt reads, single end). Reads were aligned to the mouse genome (mm10) with the BWA algorithm (default settings). Duplicate reads were removed, and only uniquely mapped reads (mapping quality ≥ 25) were used for further analysis. Alignments were extended in silico at their 3′ ends to a length of 200 bp (the average genomic fragment length in the size-selected library) and assigned to 32-nt bins along the genome. The resulting histograms (genomic “signal maps”) were stored in bigWig files. Peak locations were determined with the MACS algorithm (v2.1.0) with a cutoff P value = 10-7. Peaks that were on the ENCODE blacklist of known false ChIP-seq peaks were removed. Signal maps and peak locations were used as input data to the Active Motifs proprietary analysis program, which creates Excel tables containing detailed information on sample comparison, peak metrics, peak locations, and gene annotations.

ChIP-qPCR Chromatin obtained as described above was immunoprecipitated with 4 μg of antibodies against BACH1 (sc-271211X, Santa Cruz Biotechnology) or control IgG (I8765-10MG, Sigma-Aldrich). Samples were prepared according to the Epitect Chip OneDay kit (334471, QIAGEN). Primers used for qPCR are GPM1052127(-)01A and GPM1052368(-)01A from QIAGEN.