{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"submitter":["Shilpee Dutt"],"instrument_platform":["Illumina NovaSeq 6000"],"study_type":["RNA-seq of coding RNA"],"organism":["Homo sapiens"],"species":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-13769"],"description":["Anthracyclines, topoisomerase 2 enzyme poison that results in DNA damage, are currently used in acute myeloid leukemia (AML) treatment. Identifying the mechanisms underlying drug resistance remains an important question. Here, using a mitoxantrone-resistant cell line (HL-60/MX2), we found upregulation of DNA-PKcs, independent of the DNA damage response. We demonstrated that anthracyclines failed to induce DNA damage in resistant cells owing to the loss of expression of their target enzyme-TOP2B, rendered by DNA-PKcs directly binding to its promoter upstream region as a transcription repressor. Importantly, DNA-PKcs kinase activity inhibition re-sensitized AML relapse primary cultures and cells resistant to mitoxantrone and abrogated their tumorigenic potential in a xenograft mouse model. However, to explore other putative dysregulated pathways and genes, we performed RNA-seq experiments on HL-60/MX2 cells after siRNA-mediated knockdown of PRKDC."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Nucleic Acid Extraction - RNA was isolated from the cell pellet resuspended in TRIzol Reagent (Thermo Fisher Scientific) using the chloroform-isopropanol method. The RNA samples were quantitated on a Qubit fluorometer and appropriate dilutions were loaded on a high-sensitivity RNA screen tape to determine the RNA integrity number (RIN). Samples with 28S/18S peak integrated area ratios >2 were used for library preparation.","Library Construction - 1.5 μg of QC verified RNA samples was used for poly (A) mRNA isolation from total RNA using NEB Next Poly (A) mRNA Kit (Cat. no. NEB# 7490) according to the manufacturer’s protocol. The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (cat. no. E7760L) was used to construct double-stranded cDNA libraries from the polyA RNA fraction, using RNA fragmentation, first strand cDNA synthesis with random primers, second strand cDNA synthesis, end repair of double-stranded cDNA, adapter ligation, removal of excess adapter using sample purification beads, PCR enrichment of adapter ligated DNA, and clean-up of PCR products as instructed by the manufacturer. The cleaned libraries were quantitated on a Qubit fluorometer and appropriate dilutions were loaded on a high sensitivity D1000 screen tape to determine the size range of the fragments and the average library size.","Sequencing - Prepared libraries were sequenced on an Illumina  NovaSeq 6000 sequencer to generate 2 × 150 bp reads per sample.","Sample Collection - PRKDC knockdown: 0.2X106 cells/mL were seeded per well in a 6-well plate and transfected the next day with siRNA at 80pmols PRKDC (siRNA select-ThermoFisher, 4390824) per well using the standard 6-well protocol for Lipofectamine3000 reagent (Invitrogen, L3000015). Cells were collected 48 h post-transfection and tested for the knockdown of the respective proteins by qPCR and immunoblotting."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","MAGE-TAB Files"],"pubmed_authors":["Shilpee Dutt"],"additional_accession":[]},"is_claimable":false,"name":"RNA-seq for siPRKDC and siControl in human HL-60/MX2 mitoxantrone resistant derivative of the HL-60 cell line","description":"Anthracyclines, topoisomerase 2 enzyme poison that results in DNA damage, are currently used in acute myeloid leukemia (AML) treatment. Identifying the mechanisms underlying drug resistance remains an important question. Here, using a mitoxantrone-resistant cell line (HL-60/MX2), we found upregulation of DNA-PKcs, independent of the DNA damage response. We demonstrated that anthracyclines failed to induce DNA damage in resistant cells owing to the loss of expression of their target enzyme-TOP2B, rendered by DNA-PKcs directly binding to its promoter upstream region as a transcription repressor. Importantly, DNA-PKcs kinase activity inhibition re-sensitized AML relapse primary cultures and cells resistant to mitoxantrone and abrogated their tumorigenic potential in a xenograft mouse model. However, to explore other putative dysregulated pathways and genes, we performed RNA-seq experiments on HL-60/MX2 cells after siRNA-mediated knockdown of PRKDC.","dates":{"release":"2025-12-31T00:00:00Z","modification":"2025-12-31T02:02:11.668Z","creation":"2024-02-06T11:17:26.254Z"},"accession":"E-MTAB-13769","cross_references":{"ENA":["ERP157172"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005518","EFO_0003738","EFO_0004184"]}}