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  • br I J Kaplan Meier graph of

    2022-07-06


    (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 Doxorubicin 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).
    (legend on next page)
    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.
    (C) Western blots showing amounts of BACH1 and the loading control ACTIN in A549 cells transduced with CAS9 and sgTom or three different sgRNAs tar-geting BACH1.
    (D) Left, Transwell migration of control and BACH1-deficient A549 cells in panel C after incubation for 7 days with 1 mM NAC. Right, representative photos of migrated cells.
    (E) Western blots showing amounts of BACH1 and the loading control ACTIN in H1975 cells transduced with CAS9 and sgTom or two different BACH1 sgRNAs.
    (F) Left, Transwell migration of control and BACH1-deficient H1975 cells in panel E after incubation for 7 days with 1 mM NAC. Right, representative photos of migrated cells.
    (G) Top, weight of primary subcutaneous tumors of NGS recipient mice three weeks after subcutaneous injection of mTN-sgTom and mTN-sgBach1 cells (2.5 3 105; n = 5 mice/cell type). Bottom, number of mice having lymph node (LN) metastasis three weeks after subcutaneous injection.
    (H) Top, lung sections obtained from control and VitE-treated KP mice 8 months after intratracheal administration of pSECC-sgTom or pSECC-sgBach1 lentiviruses and stained with hematoxylin-eosin (H&E). Middle and bottom, serial sections stained with BACH1 antibody and counterstained with DAPI. The sections are representative of two mice/condition. Scale bars, 100 mm and 50 mm for the low and high magnification, respectively.
    (I) Western blots showing reduced expression of BACH1 in unselected mTC cells harvested 2 days after infection with the pSECC-sgBach1 lentivirus at a multiplicity of infection of 1. Numbers are BACH1/ACTIN-ratios as judged by densitometry.
    (J) Primary lung tumor burden of control and VitE-treated KP mice 8 months after intratracheal instillation of the control pSECC-sgTom lentivirus or the pSECC-sgBach1 lentivirus (n = 6 mice/condition).