Mycoalert assay

MDA-MB-231 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Monza, Italy) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich). 4T1 cells were grown in RPMI-1640 medium (Invitrogen) supplemented with 10% FBS. All the cells were purchased from ATCC (LGC Standards, Sesto San Giovanni, Italy). TUBO cells were cultured in DMEM supplemented with 20% FBS. Each culture medium was supplemented with penicillin (100 U/mL) and streptomycin (100 U/mL) (Sigma-Aldrich). The cells were cultured at 37 °C in a 5% CO₂ incubator and tested negative for mycoplasma using the polymerase chain reaction (PCR) MycoAlert assay (Lonza, Basel, Switzerland). Tumorspheres were generated as previously described [51 (link)]. For the spheroid generation, cells were detached with trypsin 1× (Sigma-Aldrich), washed and suspended at 20,000 cells/mL in DMEM-F12 (Invitrogen) supplemented with 10% FBS. Then, the cell suspension was mixed 1:1 in complete medium with 0.4% methylcellulose (Sigma-Aldrich) and plated at 100 µL/well in ultra-non-adherent 96-well suspension culture plates (CellStar, Greiner Bio-One, Frickenhausen, Germany).

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.

Human NSCLC cells, PC-9 (EGFRdelE746-A750), HCC827 (EGFRdelE746-A750), H1975 (EGFRL858R/T790M) and H1703 (EGFR wild-type), and immortalized non-transformed NL20 lung cells were purchased from the American Type Culture Collection. These cells tested negative for mycoplasma (Figure S8; MycoAlert assay from Lonza LT07-218) and were authenticated via DNA fingerprinting (Figure S9). These cells were maintained in either RPMI or MEM (Gibco) supplemented with 10% FBS (Hyclone), 2g/L glucose and penicillin/streptomycin (P/S). All cell lines were grown at 37°C under 5% CO₂. Cells were grown to ~80% confluence, harvested with trypsin, and suspended to the cell density required for each assay. PC-9 Gefitinib resistance (PC-9GR) was generated through chronic exposure of drug at gradual dose increments (from 50 nM up to 5 μM) for 6 months 67 (link). The IC50 of PC-9GR was determined to be about 3 μM (versus 50 nM for parental PC-9) and cells were then maintained in 1 μM of Gefitinib prior to all experiments. Further, DNA from PC-9GR were analysed to rule out the relation between the development of acquired resistance to Gefitinib of PC-9GR cells and secondary EGFR T790M mutation or MET gene amplification.

Human embryonic kidney cells (HEK293, ATCC #CRL-1573) were maintained at ~3 × 10⁶ cells per plate at 37 °C in a humidified 10% CO₂ atmosphere in Modified Eagle’s Medium (Gibco) and 10% fetal bovine serum (Invitrogen), which had been filtered through a 0.2-um PES membrane filter (Nalgene Rapid-flow). HEK293 cells were used at passage 27 to 29. Cells were collected at approximately 80 to 90% confluency. Genomic DNA of HEK293 cells was authenticated by STR profiling. Cells were confirmed to be mycoplasma-free by the Lonza MycoAlert assay.

MDA-MB-231 cells (obtained from ATCC) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) supplemented with 10% FBS and L-Glutamine (4 mmol/L) (Sigma-Aldrich) at 37 °C with 5% CO₂. The cells were cultured for less than 10 passages before experimentation and were monitored for morphological changes. STR profiling is regularly performed to authenticate the cell line using the StemElite ID Profiling Kit (Promega) at QIMR Berghofer (the last relevant test for these studies was performed February 2014, Brisbane, Australia as experiments were finalized prior to this date). Cells were tested 6-monthly for mycoplasma (MycoAlert Assay, Lonza).