biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsMarco MernbergerNextSeq 550RNA-seq of coding RNAHomo sapiensHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-13602p53 expression and transcriptional activity is repressed in human papillomavirus positive cells by the expression of viral E6 protein. E6 forms a complex with cellular E6AP protein which can lead to ubiquitination of p53, thus repressing its tumor-suppressive capabilities. In this experiment, the potential capability of a DARPin (Designed Ankyrin Repeat Protein) to reactivate p53 mediated pathways in HPV positive HeLA cells is investigated and compared to Nutlin 3a activation of p53 in HPV negative U2OS osteosarcoma cells.biostudies-arrayexpressSample Collection - HeLa and U2OS cells were transfected with pcDNA3.1(+) plasmids containing the respective DARPin using Lipofectamin 2000 transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Twenty-four hours after transfection cells were washed with PBS, detached and mRNA was isolated using the RNease mini kit (Qiagen).Nucleic Acid Extraction - RNA was isolated using the RNeasy Mini kit (Qiagen) and RNA quality was assessed using the Experion RNA StdSens Analysis Kit (Bio‐Rad).Library Construction - RNAseq libraries were prepared from total RNA with the Lexogen QuantSeq 3'-mRNA Library Prep Kit FWD for Illumina (Lexogen, 015.24) in combination with the UMI Second Strand Synthesis Module for QuantSeq FWD (Illumina, Read 1) (Lexogen, 081.96) following the manufacturer's protocol. Quality of sequencing libraries was controlled on a Bioanalyzer 2100 using the Agilent High Sensitivity DNA Kit (Agilent).Sequencing - Pooled sequencing libraries were quantified with digital PCR (QuantStudio 3D, Thermo Fisher), sequenced on the NextSeq550 platform (Illumina) with 75 base single reads.Growth Protocol - Cells were harvested and RNA was isolated using the RNeasy Mini Kit (QIAGEN, 74106) according to manufacturer’s protocol.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesJonas SchaeferVolker DötschAlexander StrubelBirgit SchäferAndreas JoergerJulianne FunkStefan KnappThorsten StiewePhilipp MünickAndreas PlückthunBirgit DreierChristian OsterburgMarco MernbergerDimitrios-Ilias BalourdasBüşra YükselMarcel TuppifalseRNAseq of HeLa and U2OS cells treated with either Nutlin, Darpin C10 targeting p53 or a control DARPinp53 expression and transcriptional activity is repressed in human papillomavirus positive cells by the expression of viral E6 protein. E6 forms a complex with cellular E6AP protein which can lead to ubiquitination of p53, thus repressing its tumor-suppressive capabilities. In this experiment, the potential capability of a DARPin (Designed Ankyrin Repeat Protein) to reactivate p53 mediated pathways in HPV positive HeLA cells is investigated and compared to Nutlin 3a activation of p53 in HPV negative U2OS osteosarcoma cells.2025-09-05T00:00:00Z2025-09-10T00:01:22.147Z2023-12-03T21:47:58.025ZE-MTAB-13602ERP155758EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003738EFO_0004184