Uw228

The human medulloblastoma cell lines DAOY and UW228 were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The ATM-deficient human fibroblast cell line AT5BIVA was obtained from the National Institute of General Medical Sciences (NIGMS). The ATM-proficient human fibroblast cell line ATCL8 was established by transfecting AT5BIVA cells with the wild-type, full-length ATM gene in a pcDNA expression vector. AT5BIVA cells were maintained in modified Eagle's medium with 20% fetal bovine serum, 100 U/penicillin, and 100 pg/mL streptomycin. DAOY and UW228 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% L-glutamine (Life Sciences) at 37°C and 5% CO₂. For transfection experiments, sub-confluent DAOY and UW228 cells were transfected in serum-free DMEM using TransIT-TKO Transfection Reagent (Mirus, Madison, WI, USA), according to the manufacturer's instructions. Short interfering RNAs (siRNA) specific for human EphB1 and the non-specific control RNAi were obtained from Invitrogen (Carlsbad, CA, USA). Briefly, cells were transfected using 10 μL TransIT-TKO for a final concentration of 25 nM siRNA for migration studies, or 50 nM siRNA for proliferation studies.

Human MB cell lines D283 and UW-228 were originally obtained from the American Type Culture Collection (ATCC, Rockville, USA). These two cell lines present molecular features of different MB molecular subgroups: UW228 cells are TP53-mutated and classified as Shh, whereas D283 cells are p53 wild-type and classified as Group ¾ (Ivanov et al., 2016 (link)). The D283 cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM low glucose, Gibco, Grand Island, USA), while UW228 cell line was cultured in DMEM: Nutrient Mixture F-12 (DMEM/F-12 Gibco®), both medium supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) and 1% (v/v) penicillin/streptomycin (Gibco). Cells were incubated in a humidified atmosphere of 5% CO₂ at 37°C.