Targeting Group3 Medulloblastoma by the anti-PRUNE-1 and anti-LSD1- KDM1A molecules augments their potential therapeutic value.
Targeting Group3 Medulloblastoma by the anti-PRUNE-1 and anti-LSD1- KDM1A molecules augments their potential therapeutic value.
Primary Group3 MB cells were plated (3x105 cells) and treated with anti-PRUNE-1 (AA7.1), anti-LSD1 (SP2577), the combination of two or vehicle (0,001% DMSO) as the negative control for the treatment. After 24 hours, the cells were lysed with TRIzol RNA Isolation Reagent (#15596018; Ambion, Thermo Fisher Scientific) and frozen at -80°C. Freshly isolated primary Group3 MB cells were collected by the biopsy after surgery resection of a patient affected by tumour (data deposited at: EVA- EMBL-EBI; project ID: PRJEB42411; analyses: ERZ1700617). These cells were cultured using Dulbecco's modified Eagle's medium (DMEM; 41966-029; Gibco) and Minimum Essential Medium (MEM; 11090081; Gibco) in a ratio 1:1 with 10% fetal bovine serum 10270-106; Gibco), 2 mM L-glutamine (25030-024; Gibco), and 1% penicillin/streptomycin (P0781; Sigma-Aldrich), with the medium changed daily.  Primary Group3 MB cells were plated (3x105 cells) and treated with anti-PRUNE-1 (AA7.1), anti-LSD1 (SP2577), the combination of two or vehicle (0,001% DMSO) as the negative control for the treatment. After 24 hours, the cells were lysed, and their RNA were extracted for RNAseq.  Total RNA, isolated from human primary medulloblastoma cells by using TRIzol RNA Isolation Reagent (#15596018; Ambion, Thermo Fisher Scientific) was quantified in a NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer (Thermo Scientific™), checked for purity and integrity. Libraries were prepared using the SMART-Seq v4 Ultra Low Input RNA for Sequencing, Nextera XT DNA Library Preparation Kit according to the protocols recommended by the manufacturer. Library kit: TSMART-Seq v4 Ultra Low Input RNA for Sequencing, Nextera XT DNA Library Preparation Kit; Library protocol: SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing User Manual, Nextera XT DNA Library Prep Kit Reference.