Nci h2452

The following mesothelioma cell lines have been used: ZL55, ZL5, ZL34, SDM103T2, SPC111 and SPC212 from our laboratory; NCI-H226, NCI-H2052, NCI-H2452, MSTO-211H^{66 (link)} were obtained from ATCC (Wesel, Germany); ACC-Meso-1 and ACC-Meso-4^{24 (link)} were obtained from Riken BRC (Ibaraki, Japan); and Mero-25, Mero-82, Mero-83, Mero-84, Mero-95^{(ref 67)} and ONE58^{(ref 68) (link)} was obtained from the European Collection of Cell Cultures (Salisbury, UK). The non-transformed mesothelial cell line SDM104^{(ref 69) (link)} was established in our laboratory. Cells established in our laboratory were maintained as described by Thurneysen et al.^{70 (link)} The rest of the cell lines were cultured in DMEM–F12 supplemented with 15% FCS and 1% Penicillin/Streptomycin solution. SV40 immortalized wild-type MEF cells and Hek 293T cells were cultured in DMEM high glucose medium supplemented with 10% FCS and 1% Penicillin/Streptomycin. All cells were cultured at 37 °C in a humidified 5% CO₂ atmosphere.

HEK293T (ATCC, no. CRL-3216), NCI-H226 (ATCC, no. CRL-5826), MSTO-211H (ATCC, no. CRL-2081), NCI-H2452 (ATCC, no. CRL-5946) mesothelioma cells, MeT-5A (ATCC, no. CRL-9444) mesothelium cells, 94T778 cells (ATCC, no. CRL-3044), 93T449 cells (ATCC, no. 3043), MIA PaCa-2 cells (ATCC, no. CRM-CRL-1420), MM.1S (ATCC, no. CRL-2974) and SKHEP-1 cells (ATCC, no. HTB-52) were obtained from American Type Culture Collection and cultured as recommended. HuCCT1 cells were gifts from Bardeesy lab in Massachusetts General Hospital. PC9 cells were gifts from Pasi lab in Dana-Farber Cancer Institute. Cells were negative for mycoplasma using MycoAlert mycoplasma detection kit (LONZA, no. LT07-418).

Human pancreatic carcinoma, PANC-1 (TKG 0606, p53 genotype: mutated), AsPC-1 (JCRB1454, null), MIA-PaCa-2 (TKG 0227, mutated) and BxPC-3 (JCRB1448, mutated) cells, and human esophageal carcinoma, TE-1 (TGK 0252, mutated at codon 272 Val to Met), TE-2 (TGK 0253, wild-type), TE-10 (TKG 0261, mutated at codon 242 Cys to Tyr), TE-11 (TKG 0262, wild-type), YES-2 (mutated at codon 236 Tyr to Asn) [19 ], YES-4 (wild-type) [20 (link)], YES-5 (mutated at codon 280 Arg to Gly) [20 (link)], YES-6 (wild-type) [20 (link)] and T.Tn (JCRB 0261, mutated at codon 214 His to Arg and 258 Glu to stop) cells were from Cell Resource Center for Biomedical Research (TKG number; Sendai, Japan), National Institutes of Biomedical Innovation, Health and Nutrition (JCRB number; Tokyo, Japan) or Dr. Yutaka Shimada (YES-2, YES-4, YES-5 and YES-6; Kyoto University, Kyoto, Japan). HEK293 cells (CRL-1573) and human mesothelioma, NCI-H2452 (CRL-5946, wild-type but truncated p53 protein), NCI-H2052 (CRL-5915, wild-type), NCI-H226 (CRL-5826, wild-type), NCI-H28 (CRL-5820, wild-type) and MSTO-211H (CRL-2081, wild-type) cells, were from ATCC (CRL number; Manassas, VA, USA). All the cells were cultured with RPMI 1640 supplemented with 10% fetal calf serum.

MSTO-211H, NCI-H2452 and 293 T cell lines (ATCC, Manassas, VA) were employed for the present study. MSTO-211H and H2452 were origin from the patients with mesothelioma and cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (Invitrogen, Carlsbad, CA), while 293 T were origin from human embryonic kidney cells and cultured in DMEM high glucose medium supplemented with 10 % fetal bovine serum. All cells were maintained in a humidified 37 °C incubator with 5 % CO₂.

Most of the pleural MM cell lines used in this study were generated as reported previously (15 (link)). Other MM lines included NCI-H2452 (catalog no. CRL-5946; RRID:CVCL_1553) and MSTO-211H (catalog no. CRL-2081; RRID:CVCL_1430), which were obtained from the ATCC. All MM cell lines were periodically screened for Mycoplasma by our Cell Culture Facility by transferring supernatant from cell cultures to indicator cultures of Vero kidney cells and then staining for cytoplasmic DNA (Mycoplasma) using Hoechst dye and fluorescence microscopy. All cell lines tested were negative for Mycoplasma contamination. Cells were maintained in RPMI1640 medium supplemented with 10% FBS containing 100 µg/mL penicillin and streptomycin and 2 mmol/L l-glutamine.

Human MPM cell lines, NCI-H28, NCI-H2052, NCI-H2596 (sarcomatoid) and NCI-H226, NCI-H2452 (epithelioid) were purchased from ATCC. Among other three epitheloid cell lines EMMeso was gifted by Dr. Tobias Peikert, Mayo Clinic and SPC111, SPC212^{23 (link)} were established by Professor Rolf A. Stahel, University of Zurich, Switzerland and gifted by Dr. Jeremy Chien, University of New Mexico. All cells were authenticated by STR profiling and crosschecked with the ATCC data bank. Cells were grown in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% antibiotic in a humidified atmosphere at 37 °C, with 5% CO₂. Virus transformed human mesothelium cell line Met-5A cells were also grown in RPMI-1640 with 10% FBS, epidermal growth factor (3.3 nM), hydrocortisone (400 nM), zinc-free bovine insulin (870 nM), and HEPES (20 mM)^{24 (link)}.

The human MPM cell lines MSTO-211H (biphasic histotype), NCI-H2452 (epithelioid histotype) and NCI-H2052 (sarcomatoid histotype) were obtained from ATCC (Manassas, VA, USA), which authenticates the phenotypes of these cell lines on a regular basis. Cells were cultured in RPMI-1640 supplemented with 10% Fetal Bovine Serum (FBS, Euroclone, Milan, Italy) and maintained at 37 °C in a water-saturated atmosphere of 5% CO₂ in air. The cell lines were routinely tested for mycoplasma contamination.