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SPANX control of Lamin A/C modulates nuclear architecture and promotes melanoma growth

ABSTRACT: Purpose: To investigate mechanisms underlying SPANX knockdown phenotypes, we monitored changes in gene expression profiles in A375 melanoma cells subjected to SPANX KD (RNA-Seq). Methods: RNASeq Libraries were prepared from isolated total RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina from Lexogen, (Vienna, Austria). Barcoded libraries were pooled and single end sequenced (1X75) on the Illumina NextSeq 500 using the High output V2.5 kit (Illumina Inc., San Diego CA). Raw data FASTQ files were generated by standstard illumina base calling pipeline and stored in illumina cloud service BaseSpace. Reads were aligned with the STASR aligner. Analyses of differentially expressed genes (DEGs) were subsequently performed using a negative binomial test method (edgeR)10 implemented using SARTools R Package Results: Of 609 genes deregulated by both shRNA, 454 were upregulated and 123 were downregulated. Networks associated with cell division were downregulated following SPANX KD, while those associated with 3-phosphoinositide biosynthesis were upregulated. Notably, IPA analysis identified the cell cycle as the primary pathway deregulated, and further analysis using the reactome database revealed that most of those genes were associated with the G1/S transition.

ORGANISM(S): Homo sapiens

PROVIDER: GSE141823 | GEO | 2020/12/31

REPOSITORIES: GEO

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Project description:Spheroids, near spherical multicellular aggregates, are one of the most common types of threedimensional (3D) cell cultures. Despite decades of implementation of spheroid technology in various fields of life science and medical research, no minimal information (MI) guidelines are available to cope with heterogeneity and to stimulate transparency. To cope with this unmet need, we assembled an international consortium to develop the MISpheroID knowledgebase (https://www.mispheroid.org) and interrogation revealed heterogeneity and lack of transparency in published spheroid-related experiments. This steered us to empirically evaluate the impact of cell line, culture medium type, spheroid formation method and spheroid size on complementary spheroid metrics (RNA fingerprints, presence of cell death, ATP content, glucose to lactate conversion, secreted protein signatures, circularity, size and cancer therapy response). We measured media-induced transcriptional variation in lung cancer (A549), colorectal cancer (HCT116), ovarium cancer (SKOV3) and glioblastoma (U87MG) spheroids using RNA sequencing (RNA-seq). These spheroids were formed in ultra-low attachment plates and cultured in 6 different media types (DMEM high glucose, DMEM/F12, RPMI1640, DMEM low glucose, EMEM and MEM) for 5 (HCT116) or 7 (A549, SKOV3 and U87MG) days. RNA extraction was performed on 2 spheroids per condition using the miRNeasy micro kit (217084, Qiagen, Hilden, Germany). RNA-sequencing libraries were prepared from purified RNA using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, Vienna, Austria) according to the manufacturer's instructions, using 27.5ng of RNA that was DNase treated using HL-dsDNA (Arcticzymes, TromsØ, Norway). The individual libraries were quantified by qPCR using the KAPA Library Quantification Kit (Roche, Pleasanton, CA, US) and equimolarly pooled. The pool concentration was measured with Qubit and 1.4pM with 1% PhiX was sequenced on a NextSeq 500 (Illumina, San Diego, CA, US) using a high-output 1x75 run. Reads were mapped to the human genome using Tophat and gene expression counts were generated using HTSeq. This data was used to perform Principal Component Analysis (PCA) and Gene Set Enrichment Analysis (GSEA).

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SPANX control of Lamin A/C modulates nuclear architecture and promotes melanoma growth

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