N ethylmaleimide

Parental NSCLC cell lines were previously described [3 (link)]. Cell lines were routinely tested and verified to be free of mycoplasma (MycoAlert Assay, Lonza). All lines were maintained in RPMI 1640 media (Hyclone or Gibco) supplemented with 5% FBS without antibiotics at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. Lenti-X 293T cells were obtained from Clontech and maintained in DMEM media (Hyclone or Gibco) supplemented with 10% FBS. Cell lines with stable expression of Streptococcus pyogenes Cas9 gene were generated by lentiviral transduction, followed by blasticidin selection as reported [59 ]. For selection of transduced cells, puromycin was added at the concentration of 1 μg/mL. Blasticidin was used at the concentration of 4 μg/mL. β-Lapachone was a gift from Professor David Boothman's lab. Auranofin (A6733-10MG) was obtained from Sigma Aldrich. N-Ethylmaleimide was purchased from Chemimpex.

Capillary fluorescent electrophoresis (CAFÉ) analysis was performed according to methods described previously by Piparia et al. and Brunn et al. (49 (link), 50 (link)). Tissue lysates and plasma samples were diluted in sample buffer (HT Pico protein express kit; Perkin-Elmer, Waltham, MA) with 10 nM N-ethylmaleimide (Chem-Impex, Inc., Wood Dale, IL) and added to 96-well skirted PCR plates (Thermo-Fisher, Waltham, MA). Dilutions were heated by using a thermal cycler (C1000 Touch; Bio-Rad, Hercules, CA) for a single 10-min cycle at 80°C and run on a LabChip GXII instrument with an HT Pico protein express chip (Perkin-Elmer, Waltham, MA). Peak area analysis was performed using WinNonlin (Pharsight, Mountain View, CA), and the total fluorescent peak area under the curve (AUC) within pertinent molecular weight ranges (2 to 16, 16 to 24, and 24 to 120 kDa) was determined and graphed using Prism software (GraphPad).