Sk n be

The human neuroblastoma cell lines SK-N-BE(1) and SK-N-BE(2) were obtained from Children’s Oncology Group (COG, USA). SK-N-BE(1) cell line was isolated from bone marrow of a 2-year-old patient when he was primary diagnosed without any treatment, and SK-N-BE(2) cell line was gained from the same patient when he has been received several courses of chemotherapy with cyclophosphamide, doxorubicin, as well as vincristine. These two cell lines had different drug sensitivity (Additional file 1: Figure S1) [11 (link)–13 (link)].
SK-N-BE (1) and SK-N-BE (2) cell lines were maintained in RPMI 1640 (Gibco, USA) with 10% fetal bovine serum (Gibco, USA), 50 units/ml penicillin, 50 μg/ml streptomycin (Yeasen, China) and 1X ITS (Sodium Pyruvate 0.11 g/L, L-glutamine 1.5 g/L, NaHCO3 1.5 g/L (Yeasen, China)). These two cell lines were incubated humidified air supplemented 5% CO2 at 37 °C.
To enable the quantitative proteomics, we used specialized stable isotope labeling with amino acids in cell culture (SILAC) medium supplemented with 10% dialyzed fetal bovine serum (Invitrogen, Darmstadt, Germany) to label the cells, where deficient Arginine and Lysine were supplemented with either stable isotope encoded heavy Arginine and Lysine (Euriso-top) or normal Arginine and Lysine for the light. Labeling efficiency was confirmed before the following quantitative full proteome analysis.

The NB cell lines used for the experiments including SK-N-BE (2 (link)), SKNDZ and SH-SY5Y were obtained from American Type Culture Collection (ATCC). Kelly cell line was kindly provided by Dr Guoliang Qing from Department of Pathophysiology, School of Basic Medical Sciences, Wuhan University. MYCN is highly expressed in SK-N-BE (2 (link)), SKNDZ and Kelly cell lines and low expressed in SH-SY5Y. All four cell lines are representative, widely used NB cell lines with relatively high feasibility. SK-N-BE (2 (link)), SKNDZ and SH-SY5Y cell lines were routinely cultured with DMEM (C11995500BT, Gibco, USA) supplemented with 10% fetal bovine serum (10099-141C, Gibco, USA) and1% Penicillin/Streptomycin (C100C5, New cell & Molecular Biotech, China), Kelly cell line was grown in complete RPMI-1640 (C11875500BT, Gibco, USA) supplemented with 10% FBS and 1% Penicillin/Streptomycin. All short tandem repeat (STR) -certified cell lines were cultured at 37C and 5% CO2 for up to 6 months after resuscitation, with mycoplasma contamination detected regularly using MycoAlert (LT07-710, Lonza, Switzerland).

Rhabdomyosarcoma Rh30 and Rh18, lung carcinoma A549 and H1299, pancreatic cancer LNCAP and PC3, neuroblastoma SKN-SH and SKN-BE cells were from American Type Culture Collection (ATCC, Rockville, MD). A549, H1299, LNCAP, PC3, SKN-SH and SKN-BE cells were cultured in DMEM (GIBCO) supplemented with 10% heat-inactivated FBS (GIBCO). Rh18 and Rh30 cells were cultured in RMPI 1640 (GIBCO) supplemented with 10% heat-inactivated FBS. 4E-BP1/2 double knock-out MEFs were from Dr. Nahum Sonenberg (McGill University, Canada), and S6K1 knock-out MEFs were provided by Dr. George Thomas (University of Cincinnati). MEF cells were cultured in DMEM supplemented with 10% heat-inactivated FBS. Control and ON-TARGETplusSMARTpool siRNAs of CDK4 were purchased from Dharmacon (Chicago, IL). Lipofectamine 2000 was from Invitrogen (Carlsbad, CA) and transfection of siRNA in cells was performed according to the manufacture's instructions.