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  • br C Western blots showing amounts of BACH and the

    2020-08-18


    (C) Western blots showing amounts of BACH1 and the loading control ACTIN in A549 Cholic acid 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).
    (K) Western blots showing increased BACH1 expression in A549 and H1975 cells transduced with SAM-sgBACH1 constructs. Control cells were transduced with a nontargeting construct (SAM-sgTom).
    (L) Scratch-wound migration assay of cells from panel J.
    (legend on next page)
    Figure S5. ChIP-Seq Analysis Identifies Known and New BACH1 Target Genes, Related to Figure 4
    (A) Known and de novo BACH1-binding motifs in mTC and mTN cells (n = 3) were identified with the findMotifGenome program of the HOMER package. The highest-ranking motif for each sample is shown.
    (B) Mean BACH1 binding to known target genes in mTC and mTN cells.
    Error bars indicate SEM.
    (legend on next page)
    Figure S6. BACH1 Is Functionally Responsible for Antioxidant-Induced Glycolysis and Migration of Mouse and Human Lung Cancer Cells, Related to Figure 5
    (A) Compensatory glycolysis in mTC and mTN cells. Values are mean of 3 cell lines/condition and normalized to values for mTC cells.
    (B) Glycolysis rates of A549 and H1975 cells transduced with SAM-sgBACH1 and control SAM-sgTom constructs.
    (C) Real-time qPCR analysis of Bach1 and BACH1-target genes in mTN cells transduced with CAS9 and either sgTom or sgBach1 (two different constructs). Data were normalized to Rplp0 expression and then to mTN-sgTom.
    (D) Western blots showing amounts of HK2 and the loading control ACTIN in mTN- sgTom and mTN-sgBach1 cells. Numbers are BACH1/ACTIN ratios as judged by densitometry.
    (G) western blots showing amounts of BACH1, HO-1, and the loading control ACTIN in H838 cells transduced with CAS9 and sgTom or two different BACH1 sgRNAs.
    (I) Real-time qPCR analysis of Bach1 and BACH1 target genes in mTC and mTN cells expressing scrambled (scr) or Bach1-targeting shRNAs. Data are mean of
    3 cell lines/condition and normalized to Rplp0 expression.
    (J) Left, scratch-wound migration assay of cells from panel I. Data are from 14 hr post-scratch. Values are the mean of 3 cell lines/condition and normalized to mTC-shScr. Right, photographs of representative scratch gaps at 14 hr.
    (K) Glycolysis rates of cells from panels I and J.
    (L) Viability of mTC and mTN cells incubated for 24 hr with 20 mM hemin.
    (M) Glycolysis rates of mTC and mTN cells incubated for 6 hr with 20 mM hemin.
    (N) Left, western blots showing amounts of BACH1 and the loading control ACTIN in human NSCLC cell lines incubated for 24 hr with 10 mM hemin. Right, amounts of BACH1 determined by densitometry; values are the mean of all seven NSCLC cell lines.
    (O) Migration of NSCLC cells incubated for 7 days with 1 mM NAC; 10 mM hemin was added at the start of the scratch-wound assay. Values are the mean of three NSCLC cell lines and normalized to untreated controls.
    (P) Glycolysis rates of NSCLC cell lines at baseline and after incubation with NAC for 7 days; 10 mM hemin was added 24 hr before Seahorse analyses. Values are the mean of six NSCLC cell lines and normalized to untreated controls.