Archives

  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • br Taqman assay for Dusp

    2020-08-18


    Taqman assay for Dusp1
    Life Technologies
    Taqman assay for Gapdh
    Life Technologies
    Taqman assay for Gclm
    Life Technologies
    Taqman assay for Hmox1
    Life Technologies
    Taqman assay for Hk1
    Life Technologies
    Taqman assay for Hk2
    Life Technologies
    Taqman assay for Mmp1a
    Life Technologies
    Taqman assay for Pkp3
    Life Technologies
    Taqman assay for Rplp0 (reference gene)
    Life Technologies
    Taqman assay for Slc16a1
    Life Technologies
    Taqman assay for Sqstm1
    Life Technologies
    Recombinant DNA
    pLKO-1 empty vector
    Dharmacon
    Dharmacon
    Dharmacon
    pGL3 Luciferase reporter vector
    Promega
    pGL3 Luciferase reporter vector positive control
    Promega
    psPAX2
    Addgene
    Addgene
    pLentiCRISPRV2blast
    Addgene
    pLentiMPHv2
    pLentiSAMv2
    pSECC Addgene, Sa´nchez-Rivera
    pWZL-Hk2-Flag
    pLPC-G6PC3
    David Bernard’s lab
    pLPCx-Grx1-ro-GFP2
    Tobias Dick’s lab
    pLPCx-ro-GFP2-Orp1
    Tobias Dick’s lab
    pCDNA-HS1
    Amit Reddi’s lab
    Software and Algorithms
    ImageJ ImageJ, NIH
    ij/index.html
    GraphPad Prism, v7
    scientific-software/prism/
    IncuCyte Zoom system
    Essen Biosciences
    Visiopharm Integrator System version 5.0.2.1158
    Visiopharm
    Seahorse Wave
    Agilent Macro excel sheet
    Agilent Seahorse
    R Statistical programming version 3.4.1 The R foundation
    http://www.r-project.org/
    (Continued on next page)
    Continued
    REAGENT or RESOURCE SOURCE IDENTIFIER
    Other
    SEEK database
    http://seek.princeton.edu N/A
    TCGA data
    http://www.cbioportal.org/ N/A
    db.org/
    Gentle MACS Dissociator
    LEAD CONTACT AND MATERIALS AVAILABILITY
    Further information and requests for resources and reagents should be directed to the Lead Contact, Martin O. Bergo ([email protected] ki.se)
    EXPERIMENTAL MODEL AND SUBJECT DETAILS
    Mice, Lung Cancer Experiments, and Antioxidant Administration
    Kras2LSL/+Trp53fl/fl mice (designated KP) (Jackson et al., 2001; Marino et al., 2000) were maintained on ML385 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, R 99% purity, Sigma) was administered in the drinking water (1 g/L). Vitamin E (DL-a-tocopheryl acetate) was administered in the chow (Lantma¨nnen) at a dose of 0.5 g/kg chow (61.5 mg/kg body weight), calculated from observed daily food intake. For allograft exper-
    iments, mouse lung tumor cells were transplanted intravenously into syngeneic mice (1 3 105 cells) or NOD-SCID-gamma mice (NSG; NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) (0.5 3 105 cells); mice were killed and lungs harvested 3 weeks later. Cells (2.5 3 105)
    were transplanted subcutaneously into NSG mice. For transplantation of human NSCLC cells, 5 3 105 cells were injected intrave-nously, 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, hydrox-ypropyl methylcellulose 0.5%) by oral gavage for 4 days. Animal experiments were approved by the Research Animal committees in Gothenburg and Linko¨ping, Sweden.
    Cell Culture and Regents
    Human NSCLC cell lines A549, H1975, H838, H1299, H23, and H460 were purchased from the American Type Culture Collec-tion (ATCC). Mouse lung tumor cells lines were isolated from Kras2LSL/+ 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% peni-cillin-streptomycin (15140122, Thermo Fisher Scientific), and 10% fetal bovine serum (FBS, 10270106, Thermo Fisher Scienti-fic). 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 ML385 in the database of commonly misidentified cell lines that is maintained by ICLAC and NCBI Biosample.