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Proteomic analysis of PHACTR1 overexpressing and knockdown HT1080 stable cell lines

ABSTRACT: The PHACTR1 gene to a range of vascular and neurological diseases, underscoring its pivotal role in biology. To investigate the role of PHACTR1 in cells, we used lentiviral transfection of HT1080 cells with plasmids to overexpress (O) or knockdown (K) the PHACTR1 gene or a scrambled control plasmid (S) and created stable cell lines. As part of a larger multi-omics study, we performed untargeted on cell lysates (n=6 per condition). Peptide extracts were fractionated into 8, and analysed by LC-MS/MS using a C18AQ column on an Ultimate nanoRSLC UPLC coupled to a Orbitrap Fusion Lumos. Mass spectrometry raw data files were searched using MaxQuant (v2.1.3.0) against the Uniprot human canonical proteome (downloaded April 2023).

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Jason C Kovacic

PROVIDER: MSV000099266 | MassIVE |

SECONDARY ACCESSION(S): PXD068755

REPOSITORIES: MassIVE

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Project description:Fanconi anemia (FA) is a genetic disorder characterized by congenital abnormalities, bone marrow failure and increased susceptibility to cancer. Of the fifteen FA proteins, Fanconi anemia group C (FANCC) is one of eight FA core complex components of the FA pathway. Unlike other FA core complex proteins, FANCC is mainly localized in the cytoplasm, where it is thought to function in apoptosis, redox regulation, cytokine signaling and other processes. Previously, we showed that regulation of FANCC involved proteolytic processing during apoptosis. To elucidate the biological significance of this proteolytic modification, we searched for molecular interacting partners of proteolytic FANCC fragments. Among the candidates obtained, the transcriptional corepressor protein C-terminal binding protein-1 (CtBP1) interacted directly with FANCC and other FA core complex proteins. Although not required for stability of the FA core complex or ubiquitin ligase activity, CtBP1 is essential for proliferation, cell survival and maintenance of chromosomal integrity. Expression profiling of CtBP1-depleted and FA-depleted cells revealed that several genes were commonly up- and down-regulated, including the Wnt antagonist Dickkopf-1 (DKK1). These findings suggest that FA and Wnt signaling via CtBP1 could share common effectors. HeLa cell line was grown in DMEM media supplemented with 10% FBS and were incubated in 5% CO2 at 37Â°C. A four-plasmid (pRSV-Rev, pMDLg/pRRE, pMD2.G and pLKO.1) expression system was used for lentiviral production. Different pLKO.1 plasmids carrying shRNAs targeting FANCA, FANCD2, CtBP1 or CtBP2 or a lentiviral control vector pLKO.1-scrambled were used. Lentiviral particles were produced via calcium phosphate-mediated transient transfection of the four plasmids into HEK293T cells. Cells were exposed to appropriate lentiviral particles. In each experiment, HeLa cells were transduced for 6 hours with filtered supernatant containing recombinant lentiviral particles. After transduction, the cells were cultured for 72 hours. Total RNA extracts from 3 different samples of each scrambled, CtBPs and FANCD2 shRNA treated HeLa cells were subjected to gene expression profiling via microarray analysis. Gene expression profiles were determined with Affymetrix GeneChipÂ® Human Gene 1.0.

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Proteomic analysis of PHACTR1 overexpressing and knockdown HT1080 stable cell lines

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