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QUANTSEQ 3' sequencing of protrusions and cell bodies of control (NT) or LARP6 siRNA (LARP6 KD) transfected MDA-MB231 breast cancer cells

ABSTRACT: QUANTSEQ 3' sequencing of protrusions and cell bodies of control (NT) or LARP6 siRNA (LARP6 KD) transfected MDA-MB231 breast cancer cells

PROVIDER: PRJEB40219 | ENA |

REPOSITORIES: ENA

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QUANTSEQ 3' sequencing of protrusions and cell bodies of control (NT) or LARP6 siRNA (LARP6 KD) transfected MDA-MB231 breast cancer cells

Project description:To assess how LARP6 affects mRNA localisation to cell-protrusions, we transfected MDA-MB231 cells with either non-targeting control siRNA or LARP6 siRNA for 72 hrs, before fractionating the cells into protrusion and cell-bodies, and analysed total RNA from each fraction via Lexogen QUANTSEQ FWD 3' mRNA sequencing. Protrusion induction was done for 2 hrs on 3 micron transwell filters (Corning). The experiment was performed twice using two independent batches of cells, and sequencing was carried out on an Illumina NextSeq 500 sequencer.

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3' mRNA-seq of protrusions and cell bodies of non-transfected (nt) or LARP6 siRNA transfected MDA-MB231 cells

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3' mRNA-seq of protrusions and cell bodies of BJ, PC-3M, RPE-1, U-87 and WM-266.4 cells

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TMT quantitative proteomics of a) protrusions and cells bodies of a panel of cell lines, b) protrusions and cells bodies of MDA-MB231 cells collected after 1 h, 2 h, 4 h, 8 h post- induction of protrusions, c) non-transfected (nt) or LARP6 siRNAs MDA-MB231 cells grown on either close or open collagen coated transwells and d) protrusions and cells bodies of a panel of breast cell lines.

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TMT analysis of LARP6 knockdown impact on protrusion induced changes in the proteome of MDA-MB231 cells

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RNAs enriched at protrusions of control or APC knockdown fibroblasts

Project description:We used a fractionation scheme to isolate protrusions and cell bodies from control or APC knockdown fibroblasts, which were induced to extend protrusions in response to addition of lysophosphatidic acid (LPA). Four replicate protrusion and cell body samples for each cell type were analyzed by RNA-Seq. To identify RNAs enriched in protrusions, a protrusion/cell body ratio was calculated. We find that ca. 7% of detected RNAs were enriched in protrusions of control cells. Knockdown of APC reduced the protrusion enrichement of a subset of these RNAs, indicating that they are dependent on APC for their localization. Localization of the remaining RNAs was not significantly affected upon APC knockdown suggesting that their localization is mediated by an APC-independent mechanism.

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Identification of mRNAS enriched in endothelial cell protrusions

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Effect of overexpressed UTR constructs on RNA enrichment at protrusions

Project description:We generated fibroblast lines overexpressing beta-globin constructs under a doxycycline-inducible promoter. These constructs contain the beta-globin coding sequence followed by either a control 3'UTR (HBB) or the Pkp4 3'UTR. The Pkp4 3'UTR carries a protrusion-targeting element and is expected to compete with localization of endogenous protrusion-localized mRNAs. We used a fractionation scheme to isolate protrusions and cell bodies from HBB and Pkp4 UTR-expressing fibroblasts, which were induced to extend protrusions in response to addition of lysophosphatidic acid (LPA). 4 replicate protrusion and cell body samples for each cell type were analyzed by RNA-Seq. To identify RNAs enriched in protrusions, a protrusion/cell body ratio was calculated. We find that ca. 4.5% of detected RNAs were enriched in protrusions of control HBB cells. Overexpression of the Pkp4 UTR reduced the protrusion enrichement of a subset of these RNAs, while the degree of localization of the remaining RNAs was not significantly affected.

2017-10-11 | GSE82230 | GEO

Analysis of LARP6 depletion impact on the proteome of MDA-MB231 breast cancer cells

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2020-10-20 | PXD021204 | Pride

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