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  • br O and P Invasion analyses of

    2020-08-18


    (O) and (P) Invasion analyses of A549 81-25-4 incubated for 14 days with NAC (O) or Trolox (P). Blue and orange dashed lines indicate cells where antioxidant administration was discontinued 48 hr before the assay.
    (Q) Table summarizing mutations in the human NSCLC cell lines used in this study.
    (S) H2O2 levels under basal conditions and 1 hr after incubation with 50 mM of the prooxidant menadione in mTC and mTN cells (n = 3/each). Values are normalized to untreated control mTC cells. (T) H2O2 levels in A549 and H838 cells incubated chronically (> 7 days) with 1 mM NAC. Values are normalized to untreated control cells. (U) Oxidation of the cytosolic H2O2 detector Orp1-roGFP2 (left) and the GSSG/GSH detector Grx1-roGFP2 (right) stably expressed in A549 cells at baseline and after menadione administration. Values are mean fluorescence measurements of 30 cells.
    (AB) Reductive potential of mTC and mTN cells displayed as GSH/GSSG ratio. Cells incubated for 24 hr with 1 mM NAC served as a positive control. Values are mean of 2 cell lines/condition and normalized to untreated mTC cells.
    (AC–AD) Migration of mTC and mTN cells incubated with 500 mM of the glutathione (GSH) synthesis inhibitor buthionine sulfoximine (BSO) (AC) and with 1 mM GSH (AD). Values are mean of 3 mTC and mTN cell lines, each assayed in 4–6 wells.
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    Figure S3. Exogenous Antioxidants Suppress the Endogenous NRF2-Dependent Antioxidant Pathway, Related to Figure 3
    (A) Gene Ontology analysis of significantly upregulated genes in mTN versus mTC lung tumor cells. Data are from RNA-seq analyses of 4 and 3 cell lines, respectively.
    (B) Heatmap showing expression of genes involved in glutathione metabolism.
    (C) Heatmap showing expression of genes from an NRF2 signature (Romero et al., 2017).
    (D) Left, western blot showing amounts of NQO1, a marker of NRF2 activity, and the loading control ACTIN in mTC and mTN cells. Right, amounts of NQO1 determined by densitometry of protein bands. n = 3 cell lines per condition.
    (E) Left, percentage of cells with nuclei staining positive for NRF2 in lung tumors of K mice (n: Ctrl = 18; NAC = 21; and VitE = 18 tumors). Tumors from all mice in Figures 1A–1C were included in the analyses. Right, photos of lung sections with representative levels of NFR2 expression in tumors of K mice. Scale bar, 50 mm.
    (F) Schematic of the current understanding of the antagonist roles of NRF2 and BACH1. Both proteins can heterodimerize with small MAF proteins and compete for binding to antioxidant response elements (ARE) motifs. In response to oxidative stress, NRF2 binds AREs, displaces repressors such as BACH1, and activates transcription of endogenous antioxidant and detoxification genes.
    (G) Kaplan-Meier graph of TCGA data showing survival of kidney cancer (KIRC) patients with high and low BACH1 expression (Z-score = 2.1).
    (H) Kaplan-Meier graph of TCGA data showing survival of pancreatic cancer (PDA) patients with high and low BACH1 expression (Z-score = 2.1).
    (I)–(J) Kaplan-Meier graph of TCGA data showing survival of LUAD patients with high and low expression of the BACH1 target genes PKP3 (I) and MMP1 (J).
    (K) Left, western blots showing amounts of BACH1 in A549 cells at baseline (Ctrl) and after incubation with 1 mM NAC or 100 mM Trolox for 7 days. ACTIN was the loading control. Right, amounts of BACH1 determined by densitometry of protein bands from six independent experiments. Aox, antioxidants.
    (L) Left, western blots of BACH1 in cytoplasmic (Cyto) and nuclear (Nucl) fractions of A549 cells incubated with 1 mM NAC for 7 days. GAPDH was the loading control for cytoplasmic fractions; LAMIN B was the loading control for nuclear fractions. Right, amounts of nuclear BACH1 determined by densitometry data from 3 cell lines in 3 independent experiments.
    (M) Western blots showing levels of NRF2, NQO1, and BACH1 in mTC and mTN cells incubated for 24 hr with 5 mM KI-696 peptide to activate NRF2.
    (N) Left, migration (Transwell assay) of mTC and mTN cells incubated for 24 hr with 5 mM KI-696. Right, representative photos of migrated cells.
    (O) Western blots showing amounts of BACH1 and ACTIN that remain in control and NAC-treated A549 cells at various times after incubation with 20 mg/ml cycloheximide (CHX).
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    Figure S4. BACH1 Mediates Antioxidant- and NRF2-Induced Migration, Mediates Antioxidant-Induced Metastasis, and Drives Migration and Metastasis in the Absence of Antioxidants, Related to Figure 4
    (A) Proliferation of mTC and mTN cells transduced with CAS9 and two different sgRNAs targeting Bach1 or a control sgRNA targeting dTomato (Tom) as judged by real-time IncuCyte analyses (n = 2 biological replicates/condition).
    (B) Left, Transwell migration of control and Bach1-deficient (sgBach1) mTC cells incubated with the NRF2-activating peptide Ki-696 (5 mM). Right, representative photos of migrated cells.