Imr 32

The human neuroblastoma SH-SY5Y and GI-ME-N cell lines were acquired from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, SK-N-AS and IMR-32 cell lines were obtained from the European Collection of Authenticated Cell Cultures (ECACC 94092302, 86041809, respectively) and the BE(2)-M17 cell line was acquired from the American Type Culture Collection (ATCC—Manassas, Virginia, United States). The SH-EP cell line was kindly provided by J. Roessler, Center for Pediatrics, Medical Center—University of Freiburg. Additional characteristics of the neuroblastoma cell lines used in this study are mentioned in Suppl. Table 1.
SH-SY5Y and BE(2)-M17 cells were maintained in 1:1 DMEM/F12 media (Gibco® Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco® Life Technologies), while GI-ME-N, SK-N-AS, SH-EP and IMR-32 cells were cultured in RPMI1640 media (Gibco® Life Technologies) supplemented with 10% FBS. Human embryonic stem cell-derived neural stem cells (hESC-NSCs) and neural crest cells (NCCs) were generated and maintained as previously described^{24 (link),59 (link),60 (link)}. No antibiotics were added to the culture media. All cell cultures were maintained at 37 °C in a 5% CO₂ humidified incubator.

Neuroblastoma cells, IMR32 (RRID:CVCL_0346), human lung fibroblasts, MRC5 (RRID:CVCL_0440), cervix epithelioid carcinoma cells, HeLa (RRID:CVCL_0030) and colon adenocarcinoma cells, LoVo (RRID:CVCL_4Y03) were purchased from ATCC (American Type Culture Collection, Manassas, VA), small cell lung carcinoma, DMS114 (RRID:CVCL_1174) were from the National Cancer Institute (NCI, New York, NY). MRC5, HeLa and LoVo cells were grown in DMEM medium, DMS114 and IMR32 were grown in RPMI‐1640 medium (Lifetechnologies Gibco, Milan, Italy). Culture medium was supplemented with 10% heat‐inactivated fetal bovine serum (Lifetechnologies Gibco, Milan, Italy), 100 units/mL penicillin and 100 μg/mL streptomycin (Lifetechnologies Gibco, Milan, Italy). All cell lines were grown at 37°C with 5% CO₂ humidified atmosphere. All experiments were performed with mycoplasma‐free cells.
All human cell lines have been authenticated using STR profiling within the last 3 years.

The human neuroblastoma cell lines IMR-32, SK-N-SH, SH-SY5Y, and SK-N-BE (2) and human embryonic kidney 293T cells were obtained from the American Type Culture Collection (Manassas, VA, United States). IMR-32, SK-N-SH, and 293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, Carlsbad, CA, United States) containing 10% fetal bovine serum (FBS, Gibco) at 37°C with 5% CO₂. SH-SY5Y and SK-N-BE (2) cells were cultured in minimum essential medium/F12 medium (Gibco) at 37°C and 5% CO₂ until reaching 70% cell density. The neuroblastoma cells were inoculated at 3 × 10⁵ cells/mL in six-well plates, with 0.5 μM doxorubicin (DOX, MedChemExpress, Monmouth Junction, NJ, United States) and 2 μM MLN8237 (MedChemExpress) added after 24 h. Two control groups [treated with complete medium and dimethyl sulfoxide (DMSO)] were established and incubated for 72 h for the cellular senescence model.

All procedures of this study were approved by the Ethics Committee of Sun Yat-sen University Cancer Center. Tissue samples and available clinical–pathological data were obtained from the Department of Pediatric Oncology, Sun Yat-sen University Cancer Center (Guangzhou, Guangdong, China) between April 2009 and June 2016, with written informed consent from participants. All cell lines, including HEK293T, SK-N-SH, SK-N-BE (2), SH-SY5Y, IMR32, and SK-N-AS cell lines were purchased from the COBIOER BIOSCIENCES (Nanjing, China). SK-N-SH and IMR32 cell lines were cultured in minimum essential medium (MEM: Gibco, USA). The SK-N-BE(2) and SH-SY5Y cell lines were cultured in MEM/F12 (1:1; MEM: Gibco, USA; F-12 basic: Gibco, USA). HEK293T and SK-N-AS cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM: Gibco, USA). Mediums of SK-N-SH, SK-N-BE (2), SH-SY5Y, and IMR32 cell lines were supplemented with 10% FBS (HyClone, USA), 1% penicillin–streptomycin solution (HyClone, USA), 1% 1 mM Sodium Pyruvate (NAP: Gibco, USA) and 1% MEM non-essential amino acids (MEM NEAA: Gibco, USA). The medium of SK-N-AS cell line was supplemented with 10% FBS (HyClone, USA), 1% penicillin–streptomycin solution (HyClone, USA), 1% non-essential amino acids (MEM NEAA: Gibco, USA). All cells were cultured in a 5% CO₂ and humidified incubator was maintained at 37 °C.