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  • br ChIP qPCR br Chromatin obtained as described


    Chromatin obtained as described above was immunoprecipitated with 4 mg of Heparin sodium 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.
    Luciferase Assays
    Gapdh and Hk2 promoter sequences (See Table S1) containing BACH1 wild-type or mutant binding sites were cloned into pGL3 Luciferase Reporter Vectors (Promega) using the restriction sites of the enzymes KpnI and HindIII. A pGL3 basic vector served as a negative control (E1751, Promega); a vector containing luciferase under the control of the SV40 promotor served as a positive control (E1741, Promega). The sequences used are shown in Table S1. Briefly, 1 3 104 cells per well of a 96-well plate were transfected with X-tremeGENE 9 DNA Transfection Reagent (6365779001, Sigma-Aldrich) at a 1:3 DNA:reagent ratio. Cells were incubated at 37 C for 48 hr and assayed as recommended by the manufacturer (Dual-Glo Luciferase Assay System, E2920,
    Promega and Viktor2, Perkin-Elmer). The values presented correspond to 5 transfections per cell line and plasmid and were normalized to total cell content determined by crystal violet staining.
    Values are presented as mean ± SEM unless stated otherwise. GraphPad Prism (v.7.0 and v.8.0) was used for statistical analyses. Cell migration and invasion curves were analyzed by two-way ANOVA. Survival was analyzed with the log-rank test. Incidences and distributions were analyzed with the chi-square test or Fisher’s exact test. A two-sided t test was used for gene expression analyses and comparisons of two groups; one-way ANOVA with Tukey’s or Dunnett’s post hoc test was used for all other variables. Experiments were repeated 2–4 times unless otherwise stated. n indicates biological replicates. P values are omitted when differences were not significant or when the n values were too low for statistical analysis.
    The accession number for the RNA-seq and the ChIP-Seq data reported in this paper is GEO: GSE128181. Individual accession
    Supplemental Figures
    (legend on next page)
    Figure S1. Antioxidants NAC and Vitamin E Increase Lung Cancer Progression and Metastasis, Related to Figure 1
    (A) Lymph node size in K mice.
    (B) Percentage of K mice with HMGA2-positive lymph nodes as judged by immunohistochemistry.
    (C) Lymph node size in KP mice.
    (D) Percentage of KP mice with HMGA2-positive lymph nodes as judged by immunohistochemistry.
    (F) Lung tumor burden (tumor area/lung area) in K and KP mice from panel E.
    (G) Hematoxylin/eosin-stained sections of lungs from K (top) and KP mice (bottom) administered NAC, VitE, or a control diet.
    (H) Representative sections of lungs from K mice showing the cytological and growth-pattern characteristics used for primary tumor grading in this study.
    (I) Left, distribution of histological grades in primary lung tumors of control and NAC- and VitE-treated K mice (n = 67, 108, and 92 tumors, respectively). Right, photos of representative tumors.
    (K) Left, percentage of Ki67-positive nuclei in lung tumors of K mice (n = 46, 76, and 82 tumors for control, NAC, and VitE groups, respectively). Right, photos of representative Ki67-stained lung sections.
    (L) Left, intensity of immunohistochemical staining for NKX2.1 in lung tumors of K mice (n = 67, 92, and 92 tumors for control, NAC, and VitE groups, respectively). Right, photos of representative NKX2.1-stained lung sections.