Geneprint 10 system kit

The genetic verification of cells was performed by analysis of STR regions of the genomic DNA, using the GenePrint 10 System kit (Promega, Madison, WI, USA). The DNA was quantified and diluted. For the pre-amplification reaction, 5 µL of Primer Pair Mix, 5 µL of Master Mix, and 10 ng of DNA in 15 µL were added to the microtube. Samples were submitted to the temperature cycles recommended by the manufacturer. PCR products were analyzed on the 3500 Genetic Analyzer (Thermo Fisher Scientific), and the sequenced fragments (containing the 9 STR loci and amelogenin) were analyzed using GeneMapper software version 4.1 (Thermo Fischer Scientific), following the recommendations of the manufacturer. Similarly, U87MG was analyzed for authenticity, and the results based on similarities were checked against the database provided by the ATCC.

H1299, HCT116, IMR90, 293T and HAFF cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% fetal bovine serum. P493-6 and MCF7 cell lines were cultured in RPMI medium 1640 containing 10% fetal bovine serum. MCF10A cell line were cultured in DMEM/F12 medium containing 5% horse serum, 20 μg/mL EGF, 0.5 μg/mL hydrocortisone, 100 ng/mL Cholera Toxin and 10 μg/mL insulin. P493-6 cells carrying a c-Myc tet-off system were provided by professor Ping Gao. All other cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were tested by STR profiling (GenePrint 10 System kit from Promega and AuthentiFiler PCR Amplification Kit from ThermoFisher) to authenticate the identity. All cells were tested for mycoplasma contamination by Cell Culture Contamination Detection Kit (ThermoFisher).

Glioma cell lines U87-MG, U251-MG, U373-MG, A172, and T98G were purchased from the ATCC (American Type Culture Collection). Patient-derived GNS166 and GNS179 cell lines were kindly provided by Steven Pollard´s Laboratory (University of Edinburgh) and grown as previously reported [49 (link)], and GB1 and GB2 were generated in the laboratory. All cell lines were mycoplasma-free and authenticated by GenePrint10System Kit (Promega, Madison, WI, USA). Glioma cell lines were cultured as adherent monolayers in Dulbecco’s modified Eagle medium (DMEM) media (cat#11995065, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Carlsbad, CA, USA), or as oncospheres in DMEM/F-12 medium (cat#10565018, Life Technologies, Carlsbad, CA, USA) supplemented with glucose 45% (Sigma-Aldrich, St. Louis, MO, USA), growth factors (20 ng/mL epidermal growth factor (EGF) and 20 ng/mL basic fibroblast growth factor (bFGF); Sigma-Aldrich), and N2 and B27 supplements (Fisher, Waltham, MA, USA). Patient-derived cell lines were also grown in this medium. Before seeding, culture plates for GNS cells were treated with 10 μg/mL of laminin (Sigma-Aldrich) for 3 h at 37  °C. Cells were maintained under standard conditions in a humidified atmosphere of 5% CO₂ at 37  °C.

Cell line identities were validated with the Promega GenePrint 10 System kit, and all experiments with the cells were performed within 1 month of their being thawed. Cells were periodically tested to confirm Mycoplasma negativity using the MycoAlert Mycoplasma Detection Kit (Lonza), at least 2 weeks before performing experiments. Cells were subcultured every 3 days. Origin and RRDI of the lymphoma cell lines are listed in Table 1.

MDA-MB-468 and HCC-38 cells, purchased from ATCC, were maintained in DMEM or RPMI medium respectively containing 10% foetal bovine serum (Invitrogen, Carlsbad, CA) and 1% antibiotics (50 μg/mL penicillin, 50 μg/mL streptomycin, 100 μg/mL neomycin), and grown at 37°C in a humidified atmosphere of 5% (v/v) CO₂ in air. This cell line was authenticated in our laboratory by using the GenePrint 10 System kit (Promega, Madison, WI, USA). We perform authentication of our cell lines each 20 passages.

All cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and authenticated every 20 passages using the GenePrint 10 System kit (Promega, Madison, WI). Cells were grown in Dulbecco’ s minimal essential medium (DMEM) or minimal essential medium (MEM), supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and 1% antibiotics (50 μg/ml penicillin, 50 μg/ml streptomycin, 100 μg/ml neomycin; Invitrogen), and maintained at 37 °C with 5% CO₂. Transfections were achieved using Lipofectamine according to the manufacturer’s instructions (Invitrogen). To generate NIH3T3 cells stably expressing VOPP1, cells were cultured for 3 weeks in a selection medium containing hygromycin (100 μg/ml). Dozens of hygromycin-resistant clones were then picked and expanded.