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Expression profiling of HCT116 cells transfected with shCTRL or shKSR1

ABSTRACT: We engineered HCT116 cells by transfecting a non targeting control hairpin or 2 different short hairpin against KSR1 to knockdown KSR1 and microarrays were performed on non-transfected controls, non-targeting shRNA controls, shRNA #1 targeting KSR1, and shRNA #2 targeting KSR1. We used microarray for molecular profiling of colon cancer cells in presence or in absence of KSR1, an important scaffold of Raf/MEK/ERK pathway and identify genes which are regulated by KSR1

ORGANISM(S): Homo sapiens

PROVIDER: GSE65351 | GEO | 2015/01/28

SECONDARY ACCESSION(S): PRJNA273753

REPOSITORIES: GEO

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Expression profiling of HCT116 cells transfected with shCTRL or shKSR1

Project description:We engineered HCT116 cells by transfecting a non targeting control hairpin or 2 different short hairpin against KSR1 to knockdown KSR1 and microarrays were performed on non-transfected controls, non-targeting shRNA controls, shRNA #1 targeting KSR1, and shRNA #2 targeting KSR1. We used microarray for molecular profiling of colon cancer cells in presence or in absence of KSR1, an important scaffold of Raf/MEK/ERK pathway and identify genes which are regulated by KSR1 Cell lines were measured in as single biological replicates 1. HCT116 untransfected 2 HCT116 + shCTRL 3.HCT116 + shKSR1#1 4. 3.HCT116 + shKSR1#2

2015-01-28 | E-GEOD-65351 | biostudies-arrayexpress

Next generation sequencing analysis of control and KSR1 knockdown CRC cell line translatomes.

Project description:KSR1 signaling alters the translational landscape of human CRC cells to support their survival. To determine the effect of KSR1 on translatomes in colon cancer cells, we performed genome-wide polysome profiling. We stably expressed short hairpin RNA (shRNA) constructs targeting KSR1 or a non-targeting control in two K-Ras mutant CRC cell lines, HCT116 and HCT15. We isolated and quantified both total mRNA and efficiently translated mRNAs (associated with ≥ 3 ribosomes) using RNA sequencing. We used Anota2seq to calculate translation efficiency (TE) by comparing the differences in efficiently translated mRNAs to the total transcript of each mRNA.

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An optimized microRNA scaffold increases shRNA processing and target knockdown

Project description:Two shRNAs were placed into expression vectors harboring mir30 microRNA scaffold and an optimized scaffold where the artificial restriction sights in mir30 have been removed. After infection and selection shRNA processing was assessed by small-RNA cloning. For both shRNAs, placement into the optimized scaffold resulted in a ~two-fold increase in processing (based on smallRNA levels). Purpose: Others have reported that the EcoRI site that was introduced to the mir30 scaffold results in decreased smallRNA processing and hence reduced target knockdown. We've developed an alternative scaffold (termed ultramir) where this site is removed. smallRNA cloning was used to determine if the movement of this sight resulted in an increase in shRNA processing. Method: Two shRNAs (one targeting Renilla Luciferase and one targeting Human RPA3) were cloned into the original mir30 cassette the ultramir cassette. Each of the 4 constructs were infected in duplicate at single copy into cells and the cells seltected unitil infection percentages reached >90% (the shRenilla hairpin was infected into HEK293T cells and the shRPA3 construts into the Gallus gallus cell line ERC. After selection smallRNA cloning was perfromed and the amount of smallRNAs corrresponding to the two shRNAs compared to the endogenous microRNA populatlon. Results: smallRNA levels of the two shRNAs doubled relative to the microRNA population when they were placed into the ultramir scaffold.

2014-11-24 | E-GEOD-62187 | biostudies-arrayexpress

Impact of small molecule and genetic inhibition of PKM2 in muscle progenitor cells

Project description:Cultured, proliferating C2C12 cells were treated with a short hairpin RNA (shRNA) targeting PKM2 or small molecules that selectively impact canonical and non-canonical PKM2 activity (shikonin and TEPP-46).

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Expression data for PAR-1-positive and -negative melanoma cell lines

Project description:PAR-1 is known to be involved in the transition from non-metastatic to metastatic melanoma. We sought to determine the downstream target genes regulated by PAR-1 to determine how PAR-1 is contributing to the metastatic melanoma phenotype. The A375SM metastatic melanoma cell line was subjected to stable transduction of lentiviral-delivered small hairpin RNA (shRNA) targeting PAR-1. As a control, a non-targeting shRNA sequence (no homology to any human gene) was also transduced into A375SM cells.

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(AP-MS) Kinase Suppressor of RAS 1 (KSR1) maintains the transformed phenotype of BRAFV600E mutant human melanoma cells

Project description:KSR1 is a scaffolding protein for the RAS-RAF-MEK-ERK pathway, which is one of the most frequently altered pathways in human cancers. Previous results have shown that KSR1 has a critical role in mutant RAS mediated transformation. Here, we examined the role of KSR1 in mutant BRAF transformation. We used CRISPR/Cas9 to knock out KSR1 in a BRAFV600E transformed melanoma cell line. KSR1 loss produced a complex phenotype characterized by impaired proliferation, cell cycle defects, decreased transformation, decreased invasive migration, increased cellular senescence, and increased apoptosis. To decipher this phenotype, we used a combination of proteomic ERK substrate profiling, global protein expression profiling, and biochemical validation assays. The results suggest that KSR1 directs ERK to phosphorylate substrates that have a critical role in ensuring cell survival. The results further indicate that KSR1 loss induces the activation of p38 Mitogen- Activated Protein Kinase (MAPK) and subsequent cell cycle aberrations and senescence. In summary, KSR1 function plays a key role in oncogenic BRAF transformation.

2023-10-24 | PXD036261 | Pride

(Full Proteome) Kinase Suppressor of RAS 1 (KSR1) maintains the transformed phenotype of BRAFV600E mutant human melanoma cells

Project description:KSR1 is a scaffolding protein for the RAS-RAF-MEK-ERK pathway, which is one of the most frequently altered pathways in human cancers. Previous results have shown that KSR1 has a critical role in mutant RAS mediated transformation. Here, we examined the role of KSR1 in mutant BRAF transformation. We used CRISPR/Cas9 to knock out KSR1 in a BRAFV600E transformed melanoma cell line. KSR1 loss produced a complex phenotype characterized by impaired proliferation, cell cycle defects, decreased transformation, decreased invasive migration, increased cellular senescence, and increased apoptosis. To decipher this phenotype, we used a combination of proteomic ERK substrate profiling, global protein expression profiling, and biochemical validation assays. The results suggest that KSR1 directs ERK to phosphorylate substrates that have a critical role in ensuring cell survival. The results furtherindicate that KSR1 loss induces the activation of p38 Mitogen-Activated Protein Kinase (MAPK) and subsequent cell cycle aberrations and senescence. In summary, KSR1 function plays a key role in oncogenic BRAF transformation.

2023-08-15 | PXD036265 | Pride

T47D shNT versus T47D shRON

Project description:Introduction: HGFL-Ron signaling is augmented in human breast cancer and is associated with poor overall prognosis. Here, we investigate the role of HGFL-Ron signaling in RON-modulated human luminal A breast cancer cells T47D through RNA-sequencing, focusing on the impact of Ron knockdown through a short hairpin construct. Methods: non-targeting shRNA (shNT) and RON-targeting shRNA (shRON) were submitted for transcriptomic characterization on the Illumina HiSeq 2500. High quality reads were aligned to the hg19 genome and quantified to generate RPKM

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mRNA sequencing to assess RfxCasR and matching shRNA specificity

Project description:Matching sets of RfxCasR and shRNAs targeting ANXA4 and B4GALNT1 plus non-targeting (NT) controls were profiled by mRNA sequencing to compare non-specific transcriptome perturbations for both shRNA and RfxCasR technologies.

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Methylated RNA immunoprecipitation sequencing in the Kasumi-1 cell line transfected with WTAP shRNA or scramble control shRNA

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