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Analysis of Nucleolin depletion impact on the KRAS-induced proteomic changes in mouse Pancreatic ductal adenocarcinoma cells.

ABSTRACT: To reveal the role of Nuclelin (Ncl) downstream of mutant KRAS in shaping the proteome of Pancreatic Ductal Adenocarcinoma (PDAC) cells, we carried out a quantitative proteomics 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). Tandem Mass Tagging (TMT) was employed for quantitative analysis of Dox induced (24hrs) vs. non-induced iKras-PDAC cells, from control or Nucleolin depleted settings. Two independent siRNA oligos were used against Ncl. All samples barcoded and pooled together using TMT 6plex labelling kit (Thermo).

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

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

DISEASE(S): Carcinoma

SUBMITTER: Faraz Mardakheh

LAB HEAD: Faraz K. Mardakheh

PROVIDER: PXD038338 | Pride | 2023-02-12

REPOSITORIES: Pride

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			Action	DRS
	QE2_20200611_NclKD_1_F1.raw	Raw
	QE2_20200611_NclKD_1_F2.raw	Raw
	QE2_20200611_NclKD_1_F3.raw	Raw
	QE2_20200611_NclKD_1_F4.raw	Raw
	QE2_20200611_NclKD_1_F5.raw	Raw

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Analysis of the impact of Nucleolin phospho-mutants on the proteome of pancreatic ductal adenocarcinoma cells.

Project description:To reveal the impact of Nucleolin (Ncl) phosphorylation on oncogenic KRAS-induced changes in the proteome of Pancreatic Ductal Adenocarcinoma (PDAC) cells, we carried out a quantitative proteomic analysis on tumour cells isolated from an inducible mouse model of PDAC (iKras PDAC) (Ying et al., 2012), which were ectopically expressing wild-type or different phospho-mutants of Ncl. In this mouse model, oncogenic Kras (G12D) expression can be controlled by administration of doxycycline (Dox). Tandem Mass Tagging (TMT) was employed for quantitative analysis of Dox induced (24hrs) vs. non-induced iKras-PDAC cells, which were privously transfected with three different expression constructs of mouse myc-tagged Ncl. These constructs encoded for wild-type Ncl (WT Ncl), a phospho-defective mutant with four Serine to Alanine mutations on S28, S34, S41, and S42 sites (S4A Ncl), and a phospho-mimicking mutant with the same Serines mutated to Aspartic Acid (S4D Ncl). Cells were subjected to whole cell lysis, followed by Trypsin digestion, TMT barcoding with TMT 6plex kit (Thermo), and peptide fractionation, before analysis by LC-MS/MS.

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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 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

Analysis of oncogenic KRAS impact on the phospho-proteome of PDAC cells.

Project description:To reveal the impact of mutant KRAS on the phospho-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). Tandem Mass Tagging (TMT) was employed for quantitative analysis of Dox induced (24hrs) vs. non-induced iKras-PDAC cells. To assess the impact of ERK1/2 signalling, we also analysed cells induced with Dox in presence of Trametinib (10nM), a potent and specific inhibitor of ERK1/2 signalling. A total of 3 biological replicates per condition were assessed, with all samples barcoded and pooled together using TMT 10plex labelling kit (Thermo). To enrich for phospho-peptides, TiO phospho-peptide enrichment was performed. Both the phospho-enriched as well as the total peptide pools were analysed by mass spectrometry and the phopho to total ratio was calculated as a specific measure of phosphorylation change.

2023-02-12 | PXD030893 | 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

Analysis of oncogenic Kras-induced changes in the RNA-bound proteome of PDAC cells

Project description:In order to reveal the impact of oncogenic KRAS signaling on the RNA-bound proteome of Pancreatic Ductal Adenocarcinoma (PDAC), we carried out quantitative whole Transcriptome RNA Interactome Capture (RIC) analysis in tumour cells 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). For RIC, we employed Orthogonal Organic Phase Separation (OOPS)(Queiroz et al., 2019). In this method, UV-C crosslinking coupled with phenol-chloroform based phase separation is used to isolate RNA-crosslinked proteins, which concentrate in the interface fraction, whilst free proteins concentrate in the organic fraction. SILAC was employed for quantitative analysis of Dox induced (24hrs) changes in the RNA-bound proteome. To assess the impact of ERK signalling, an independent experiment was carried out in which Trametinib (10nM) was added to the cells at the same time as Dox (both with or without Dox cells). A total of 6 biological replicates without Trametinib, and 4 biological replicates with Trametinib, were conducted, with switching the Heavy and Light SILAC labels for +Dox and -Dox conditions. Both the Interface as well as the organic phase fractions were analysed by mass spectrometry. In a separate experiment, the total proteome changes in response to dox (24hrs) were quantified from two reciprocally SILAC labelled mixes of heavy and light iKras PDAC whole cell lysates (See the READ ME.XLS for details of the experimental conditions).

2023-02-12 | PXD030825 | Pride

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Project description:We constructed a primary lung cell model to permit regulated expression of KRASG12D. To do this, we leveraged a non-transformed, immortalized, human primary bronchial epithelial cell line (HBEC; hTert, CDK4, TP53 knockdown) that remains anchorage dependent and do not develop tumors when implanted into mice. We next modified these cells by stably integrating a regulatable KRASG12D allele, iKRASG12D, such that physiological expression of mutant KRAS is activated upon addition of doxycycline. The HBEC-iKRAS (WT) cell line and HBEC-iKRASG12D (MUT) cell line were propagated with or without Doxycycline (500ng/ml) respectively. RNA profiling of HBEC-iKRASG12D and HBEC-iKRASWT cells revealed widespread changes for HBECs harboring the activated KRAS allele in the presence of Dox. Within the KRASG12D-induced genes, the Molecular Signatures Database identified the oncogenic RAS signature as a top-enriched gene set. Upregulation of Ras signaling in Dox-treated HBEC-iKRASG12D cells was also supported by a significant overlap with a KRAS signature previously characterized by Singh et al.

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