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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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			Action	DRS
	20200821_opencloselarp6KD_1_A_R1.raw	Raw
	20200821_opencloselarp6KD_1_B_R1.raw	Raw
	20200821_opencloselarp6KD_1_C_R1.raw	Raw
	20200821_opencloselarp6KD_1_D_R1.raw	Raw
	20200821_opencloselarp6KD_1_E_R1.raw	Raw

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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

Quantitative analysis of the impact of CX-5461 on the proteome of Pancreatic ductal adenocarcinoma cells.

Project description:To reveal the effect of CX-5461 (a small-molecule inhibitor of ribosome biogenesis)on the proteome of Pancreatic Ductal Adenocarcinoma (PDAC) cells, we carried out two quantitative proteomics analyses, using tumour cells isolated from an inducible mouse model of PDAC (iKras PDAC) (Ying et al., 2012). In this model, oncogenic Kras (G12D) expression can be controlled by administration of doxycycline (Dox). In the first experiment, Tandem Mass Tagging (TMT) was employed for quantitative analysis of mock vs. CX-5461 (100nM) treated iKras cells for 48 hrs, in presence of Dox (Kras on). In the second experiment, TMT was used to quantitatively analyse the proteomics changes induced by Dox removal (48 hrs), in presence or absence of CX-5461. For this purpose, iKras PDAC cells were seeded and grown for 48 hrs with or without Dox, in the background of mock vs. CX-5461 (100nM) co-treatments. TMT labelling was done using the TMT 6plex labelling kit from Thermo Fisher.

2023-02-12 | PXD038439 | Pride

Quantitative timecourse analysis of oncogenic KRAS expression impact on the proteme of pancreatic ductal adenocarcinoma cells.

Project description:To reveal the impact of mutant KRAS expression on the proteome of Pancreatic Ductal Adenocarcinoma (PDAC) cells, we carried out a timecourse quantitative proteomic analysis of tumour cells isolated from an inducible mouse model of PDAC (iKras PDAC) (Ying et al., 2012). In this model, oncogenic Kras (G12D) expression can be controlled by administration of doxycycline(Dox). Cells were grown in the absence of Dox for 48 hrs, before being treated with or without Dox for 4, 8, 12, 24, and 36 hrs, followed by total lysis and quantitative proteomics analysis using Tandem Mass Tagging (TMT). A total of 2 biological replicate experiments were analysed, with all samples from each replicate barcoded and pooled together using TMT 10plex labelling kit (Thermo).

2023-02-12 | PXD038440 | Pride

Quantitative timecourse proteomics analysis of oncogenic KRAS removal in mouse Pancreatic Ductal Adenocarcinoma Cells

Project description:To reveal the impact of mutant KRAS on the proteome of Pancreatic Ductal Adenocarcinoma (PDAC) cells, we carried out a quantitative phospho-proteomic analysis of tumour cells isolated from an inducible mouse model of PDAC (iKras PDAC) (Ying et al., 2012). In this model, oncogenic Kras (G12D) expression can be controlled by administration of doxycycline (Dox). A timecourse Dox removal experiment was carried out in which cells with or without Dox removal at 12, 24, 36, and 48 hrs intervals were lysed and analysed by quantitative proteomics using Tandem Mass Tagging (TMT). Two independent biological replicate experiments were carried out, with timecourse samples in each replicate being barcoded and pooled together using TMT 10plex labelling kit (Thermo).

2023-02-12 | PXD038441 | 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

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

Project description:We analysed the impact of LARP6 depletion on the proteome of actively growing MDA-MB231 breast cancer cells by SILAC. For this purpose, Light (L) SILAC-labelled MDA-MB231 cells were treated with non-targeting control (NT) or two independent LARP6 siRNA (18i & 97i) for 72 hrs, before lysis in 4% SDS, 100mM Tris/HCl pH 7.5. In parallel, Heavy (H)SILAC labelled non-transfected MDA-MB231 cells were grown and lysed similarly. Each L labeled lysate was then mixed with an equal amount of H labelled lysate. Mixing of samples to the same H standard therefore allowed cross-comparison of different siRNA treatments from separate runs.

2020-10-20 | PXD021204 | 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 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

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