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

ABSTRACT: To assess if LARP6 is required for upregulation of ribosomal proteins upon protrusion induction, MDA-MB231 breast cancer cells were treated with non-targeting (NT) or LARP6 siRNA for 48 hrs, before being seeded on top of collagen-I coated 3 micron transwell filters (corning). No media was added to the bottom chamber and cells were allowed to adhere to filter overnight. The next day, the media on top of cells was refreshed, and the filters were either left with no media in the bottom chamber (closed pores), or with media also added to the bottom chamber (open pores) in order to allow formation of protrusions. Cells were allowed to form protrusions for 12 hrs before being lysed and analysed by mass spectrometry.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Maria Dermit Salazar

LAB HEAD: Faraz Mardakheh

PROVIDER: PXD021180 | Pride | 2020-10-20

REPOSITORIES: Pride

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Quantitative proteomics analysis of actin-rich protrusions and cell-bodies in migratory cells

Project description:This upload concerns 2 independent but related experiments that were searched together: a) To reveal proteins that consistently localise to actin-rich protrusions across different human migratory cell-lines, we profiled the distribution of cellular proteins between protrusions and cell-bodies in a panel of 5 established human cell-lines (see experimental design). Cell lines were seeded on top of collagen-I coated 3 micron transwell filters (corning). No media was added to the bottom chamber and cells were allowed to adhere to the filter overnight. The next day, the media on top of the cells was refreshed, and media was also added to the bottom chamber in order to open the pores and allow formation of protrusions. Cells were allowed to form protrusions for 2 hrs before being fixed with ice-cold methanol. Cells were then rehydrated in PBS, followed by independent lysis of protrusions and cell-bodies on opposite sides of the filters in 2% SDS, 100mM Tris pH 7.5 b) To analyse the temporal dynamics of protein localisation to cell protrusions, we carried out a time-course spatial proteomics analysis of protrusions and cell-bodies in MDA-MB231 cells. Cells were seeded on top of collagen-I coated 3 micron transwell filters (corning). No media was added to the bottom chamber and cells were allowed to adhere to filter overnight. The next day, the media on top of cells was refreshed, and the filters were either left with no media in the bottom chamber (closed pores), or media added to the bottom chamber (open pores) for different lengths of times (1, 2, 4 , & 8 hrs) to induce protrusion formation. Cells were then fixed with ice-cold methanol, rehydrated in PBS, followed by independent lysis of protrusions and cell-bodies on opposite sides of each filter by 2% SDS, 100mM Tris pH 7.5.

2020-10-20 | PXD021239 | Pride

Assessing the short and long-term impacts of protrusion induction on the proteome of MDA-MB231 cells

Project description:To assess the impact of protrusion induction on the proteome of mesenchymal-like migratory cells, MDA-MB231 breast cancer cells were seeded on top of collagen-I coated 3 micron transwell filters (corning). No media was added to the bottom chamber and cells were allowed to adhere to filter overnight. The next day, the media on top of cells was refreshed, and the filters were either left with no media in the bottom chamber (closed pores), or with media also added to the bottom chamber (open pores) in order to allow formation of protrusions. Cells were allowed to form protrusions for 2 or 24 hrs, before being lysed and analysed by mass spectrometry, using TMT mediated quantitative proteomics.

2020-10-20 | PXD021206 | Pride

Assessment of nascent protein accumulation in different subcellular fractions following protrusion induction

Project description:To assess if newly synthesised ribosomal proteins participate in canonical ribosome biogenesis in the nucleus, following their enhanced translation upon protrusion induction, Light (L) SILAC labelled MDA-MB231 breast cancer cells were seeded on top of collagen-I coated 3 micron transwell filters (corning). No media was added to the bottom chamber and cells were allowed to adhere to filter overnight. The next day, the media on top of cells was replaced with either medium (M) (closed pores) or heavy (H) SILAC media to pulse label newly synthesised proteins. H media was also added to the bottom chamber (open pores) in order to allow formation of protrusions in the H condition. Cells were pulsed for 4 hrs before being lysed and H and M pulsed samples were mixed together. Mixed lysates were then subjected to subcellular fractionation by serial solubilization (cytosol, membrane and nucleus) using Pierce subcellular protein fractionation kit (78840).

2020-10-20 | PXD021205 | Pride

Pulsed-SILAC analysis of protein translation rate changes upon protrusion formation

Project description:To assess the impact of protrusion formation on protein synthesis rates on a global scale, we carried out a pulsed-SILAC (pSILAC) analysis in MDA-MB231 cells grown with or without protrusions for 1, 2, 4, and 8 hrs. Briefly, light SILAC labelled cells were seeded on top of 2x 3 micron transwell filters without any media added to the bottom chamber. Cells were allowed to attach to the filter overnight, and the next day the media on the top was changed to medium (M) or heavy(H) SILAC DMEM. At the same time, the H SILAC media was also added to the bottom chamber of the transwell with H media on the top to induce protrusions. Cells were then allowed to form protrusions for 1, 2, 4, or 8 hrs, before being lysed and mixed with the M labelled lysates (cells without protrusions) from the same timepoint. The experiment was then repeated with switched SILAC labelling.

2020-10-20 | PXD021203 | Pride

Protrusion vs cell-body mRNA analysis by RNA-seq

Project description:We quantified the distribution of cellular mRNAs between protrusions and cell-body of MDA-MB-231 breast carcinoma cells by RNA-seq, using a filter based fractionation method. In this method, cells are seeded on top of 3micrometer transwell filters to enable protrusions to form but to prevent the cell-bodies passing through due to the small size of the pores, thus resulting in separation of protrusions and cell-bodies on opposite sides of the filter, from which RNA can be prepared separately. RNA samples for Illumina sequencing were generated in duplicates from two biological replicate experiments (two protrusions and two cell-body).

2015-11-08 | E-MTAB-2546 | biostudies-arrayexpress

Protrusion vs Cell-body SILAC proteomics

Project description:In mesenchymal-like cell migration, cells need to polarise into a protrusive front and a retracting cell body. To understand this process better, we set out to quantify the distribution of cellular proteins between protrusions and cell-body by proteomics, using MDA-MB-231 cells, a highly invasive breast cancer cell-line. We utilised a transwell filter based fractionation method in conjugation with SILAC proteomics. In this method, cells are seeded on top of 3μm transwell filters to enable protrusions to form through the pores of the filters but to prevent the cell-bodies passing through due to the small size of the pores, thus resulting in separation of protrusions and cell-bodies on opposite sides of the filter, which can then be lysed and prepared separately. Prepration of protrusion and cell-body fractions from heavy and light SILAC labelled cells then allows for reciprocal mixing and quantification of proteins between protrusions and cell-body. In this study, we determined the relative distribution of 3240 proteins between protrusions and cell-body from two SILAC mixes. Associated ArrayExpress data: E-MTAB-2546.

2015-10-09 | PXD000914 | Pride

Protrusion vs Cell-body pulsed-SILAC proteomics

Project description:In mesenchymal-like cell migration, cells need to polarise into a protrusive front and a retracting cell body. Apart from proteins, several mRNAs are known to be localised to cell protrusions but to what extent RNA localisation and local translation contributes toward front-back assymetry is unknown. We set out to quantify the relative translation rates of cellular proteins between protrusions and cell-body by Pulsed-SILAC proteomics, using MDA-MB-231 cells, a highly invasive breast cancer cell-line. We utilised a transwell filter based fractionation method in conjugation with pulsed-SILAC. In this method, cells are seeded on top of 3μm transwell filters to enable protrusions to form through the pores of the filters but to prevent the cell-bodies passing through due to the small size of the pores, thus resulting in separation of protrusions and cell-bodies on opposite sides of the filter, which can then be lysed and prepared separately. Prepration of protrusion and cell-body fractions from SILAC heavy vs. medium label pulsed cells then allows for reciprocal mixing and quantification of nascent proteins between protrusions and cell-body. We determined the relative local translation rates of 1150 proteins between protrusions and cell-body from two pulsed-SILAC mixes.

2015-10-09 | PXD002649 | Pride

Protrusion vs Cell-body SILAC proteomics

Project description:In mesenchymal-like cell migration, cells need to polarise into a protrusive front and a retracting cell body. To understand this process better, we set out to quantify the distribution of cellular proteins between protrusions and cell-body by proteomics, using MDA-MB-231 cells, a highly invasive breast cancer cell-line. We utilised a transwell filter based fractionation method in conjugation with SILAC proteomics. In this method, cells are seeded on top of 3Î¼m transwell filters to enable protrusions to form through the pores of the filters but to prevent the cell-bodies passing through due to the small size of the pores, thus resulting in separation of protrusions and cell-bodies on opposite sides of the filter, which can then be lysed and prepared separately. Prepration of protrusion and cell-body fractions from heavy and light SILAC labelled cells then allows for reciprocal mixing and quantification of proteins between protrusions and cell-body. In this study, we determined the relative distribution of 3240 proteins between protrusions and cell-body from two SILAC mixes. Associated ArrayExpress data: E-MTAB-2546.

2016-03-10 | MSV000079570 | MassIVE

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.

2020-10-30 | E-MTAB-9520 | biostudies-arrayexpress

Analysis of RNA distribution between pseudopodia and cell bodies in response to Fibronectin

Project description:The goal of the study was to identify on a genome-wide scale RNAs that are enriched at the leading edge of migrating cells. For this, we employed a fractionation method in which cells are plated on a microporous filter whose bottom side only is coated with fibronectin. The cells thus polarize and extend pseudopodial protrusions towards the bottom surface. These protruding pseudopodia can then be physically isolated from the bottom surface of the filter and their contents compared with the remaining cell bodies, which are isolated from the upper surface of the filter. Keywords: comparative RNA distribution

2008-03-18 | GSE10227 | GEO

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