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5fa517c9-67f8-4ec7-88bd-08a5a4c833c5 - samples

ABSTRACT: In this study, we profiled single-cell transcriptome (10X genomics) of Patient-derived xenografts (PDX) T-ALL replase samples from P1 patient. Primary human T-ALL cells were recovered from cryopreserved bone marrow aspirates of patients enrolled in the ALL-BFM 2009 study. Patient-derived xenografts (PDX) were generated as previously described by intrafemoral injection of 1 Million viable primary ALL cells in NSG mice110 PDX-derived (P1)28 cells were frozen until processing. For scRNA-seq library preparation, cryopreserved cells were thawed rapidly at 37 Ã¢Â„Âƒ and resuspended in 10 ml warm Roswell Park Memorial Institute (RPMI) medium with 100 ÃŽÂ¼g/ml Dnase I. Cells were centrifuged for 5 mins at 300 g, and resuspended in ice-cold phosphate buffered saline (PBS) with 2% foetal bovine serum (FBS) and 5mM EDTA. Cells were stained on ice with anti-murine-CD45-PE (mCD45)(clone 30-F11; BioLegend; 1:20) in the dark for 30 mins. 1:100 DAPI was added and incubated in the dark for 5 mins before sorting. Triple negative cells (DAPI-mCD45-GFP-) were sorted (Fig. S27) using a BD FACSAriaÃ¢Â„Â¢ Fusion Cell Sorter into ice cold 0.03% bovine serum albumin (BSA) in PBS. All isolated cells were immediately used for scRNA-seq libraries, which were generated as per the standard 10x Genomics Chromium 3Ã¢Â€Â² (v.3.1 Chemistry) protocol. Completed libraries were sequenced on a NextSeq5000 sequencer (HIGH-mode, 75 bp paired-end).

PROVIDER: EGAD00001008325 | EGA |

REPOSITORIES: EGA

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Project description:High activation of the PI3K-AKT-mTOR pathway is characteristic for T-cell acute lymphoblastic leukemia (T-ALL). Here we investigated how 4EGI-1, a small inhibitor of cap-dependent translation, induces apoptosis in T-ALL clones displaying hyperactive PI3K-AKT-mTOR signaling. 4EGI-1 treatment reduced the ribosomal occupancy of transcripts that define the molecular difference between T-ALL blasts and normal thymocytes. These transcripts encoded proteins for the translational machinery, for mitochondrial metabolism as well as oncoproteins such as Cyclin D1, Bcl-2 and c-myc. Therefore, targeting translation initiation proves valuable in situations where mTOR is activated by multiple events. For each biological replicate 5 x 107 #483 T-ALL cells were treated with 100 ÂµM 4EGI-1 or DMSO. After 2 h incubation at 37Â°C cells were washed in ice-cold PBS containing cycloheximide (0.1 mg/ml), pelleted and resuspended in extraction buffer (20 mM Tris-HCl, pH 8.0, 140 mM KCl, 0.5 mM DTT, 5 mM MgCl2, 0.5 % nonidet-P40, 0.1 mg/ml cycloheximide, 10000 IU/ml dalteparin sodium) and incubated for 5 min on ice. All subsequent steps were carried out in the cold. After centrifugation for 10 min at 12000 x g, 0.4 ml supernatant was layered onto a 12 ml linear sucrose gradient (10 - 50% sucrose [w/v] in extraction buffer without nonidet-P40) and centrifuged at 4 Â°C in an SW40Ti rotor (Beckman) at 35000 rpm without brake for 120 min. The gradients were harvested and absorbance profiles at 254 nm recorded (type 11 optical unit/UA-6 detector, ISCO). 1 ml fractions were collected and supplied with 0.1 volume of 3 M sodium acetate (pH 5.2) and 1 volume of isopropanol for overnight precipitation at -20 Â°C. RNA was purified using Nucleospin RNAII tubes (Macherey & Nagel) following the manufacturerÂ´s protocol. For microarray analysis RNA from polysome fractions were pooled, precipitated by adding LiCl to a final concentration of 2 M and resolubilized in 15 Âµl of RNase-free water. RNA concentrations were determined and the samples were stored at -80 Â°C.

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5fa517c9-67f8-4ec7-88bd-08a5a4c833c5 - samples

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