ABSTRACT: Ba/F3 cells were transformed after transfection with CRISPR/CAS9 + gRNA vs target gene. Oligoclonal cell population was flow sorted into single cells and processes for RNAseq.
Project description:Transcriptional profiling of transformed Ba/F3 cells by myeloproliferative neoplasm-associated JAK2 V617F mutant comparing control Ba/F3 cells expressing wild type JAK2. Two-condition experiment, WT cells vs. VF cells. One replicate per array.
Project description:The identification and functional validation of potentially oncogenic mutations in leukemia is an essential step toward a future of personalized targeted therapy. To assess the oncogenic capacity of individual mutations, reliable and scalable in vitro experimental approaches are required. Since 1988, researchers have used the IL-3 dependent Ba/F3 transformation assay to validate the oncogenic potential of mutations to drive factor-independent growth. Here we report a previously unrecognized phenomenon whereby Ba/F3 cells, engineered to express weakly transforming mutations, present with additional acquired mutations in the expressed transgene following factor withdrawal. Using four mutations with known transformative capacity in three cytokine receptors (CSF2RB, CSF3R and IL7R), we demonstrate that the mutated receptors are highly susceptible to acquiring additional mutations. These acquired mutations of unknown functional significance are selected by factor withdrawal but appear to exist prior to the removal of growth factor. This anomaly has the potential to confound efforts to both validate and characterize oncogenic mutations in leukemia, particularly when it is not standard practice to sequence validate cDNAs from transformed Ba/F3 lines. We present specific recommendations to detect and mitigate this phenomenon in future research using Ba/F3 transformation assays, along with methods to make the Ba/F3 assay more quantitative.
Project description:Paxillin is a focal adhesion adaptor protein, heavily phosphorylated at multiple tyrosine residues, as well as at serine 273 (S273), and is known to be critical for cytoskeleton rearrangement and cell migration. We previously found that paxillin plays a regulatory role in IL-3-dependent survival of Ba/F3 cells, a mouse pro-B cell line. In this study, by using overexpressed His6 tagged-paxillin as a bait, we found that DDX42, a DEAD-box RNA helicase, interacted with paxillin, inhibited apoptosis, and promoted polarization of Ba/F3 cells. His6 tagged-paxillin was stably overexpressed in Ba/F3 cells, pulled-down from cell lysates with Ni+-NTA beads, and analyzed by one-dimensional SDS-PAGE followed by LC-MS. We found that DDX42 co-precipitated with paxillin, as demonstrated by western blotting analysis of His6 tagged-paxillin precipitates with anti-DDX42 antibodies and His6 tagged-DDX42 precipitates with anti-paxillin antibodies. In addition, we observed a preferential interaction of DDX42 with the paxillin mutant, S273A, compared to the S273D mutant. Furthermore, DDX42 overexpression in Ba/F3 cells delayed the apoptosis induced by IL-3 deprivation and promoted restoration of the elongated shape in Ba/F3 cells induced by IL-3 re-supply after a 6 h-deprivation. These results suggested that DDX42 interacts with paxillin and participates in IL-3-dependent cell survival, as well as in the cytoskeletal rearrangements underlying polarization of Ba/F3 cells.
Project description:Gene expression profiles in Ba/F3 cells expressing ETV6-PDGFRB, FIP1L1-PDGFRA or a control vector, treated or not with imatinib (Glivec) Ba/F3 cells expressing FIP1L1-PDGFRA or ETV6-PDGFRB were cultured in the presence or absence of imatinib for 4 hours before RNA extraction followed by hybridization on Affymetrix microarrays. In a control condition Ba/F3 cells were cultured in the presence of IL3 in the absence or in the presence of imatinib for 4 hours before RNA extraction. 4 hours treatment with imatinib in Ba/F3 cells expressing ETV6-PDGFRB, FIP1L1-PDGFRA or a control vector
Project description:Gene expression profiles in Ba/F3 cells expressing ETV6-PDGFRB, FIP1L1-PDGFRA or a control vector, treated or not with imatinib (Glivec) Overall design: Ba/F3 cells expressing FIP1L1-PDGFRA or ETV6-PDGFRB were cultured in the presence or absence of imatinib for 4 hours before RNA extraction followed by hybridization on Affymetrix microarrays. In a control condition Ba/F3 cells were cultured in the presence of IL3 in the absence or in the presence of imatinib for 4 hours before RNA extraction. 4 hours treatment with imatinib in Ba/F3 cells expressing ETV6-PDGFRB, FIP1L1-PDGFRA or a control vector
Project description:The aim of the study is to analyse whether the Sorafenib renders FLT3-ITD-positive acute myeloid leukemia (AML) cells more immunogenic . We used Ba/F3-ITD cells as a model cell line to study the effect of Sorafenib on FLT3-ITD-positive AML cells. Ba/F3-ITD cells are murine pro-B cell lines with a stable FLT3-ITD expression. Ba/F3-ITD cells were treated with DMSO or 10nM sorafenib for 24 hours. Cells were harvested and total RNA was isolated
Project description:Taking a series of oncogenic protein tyrosine kinases and constitutively expressing them in Ba/F3 cells, proteomic analysis was utilised in order to identify common protein changes.
Project description:The aim of the study is to analyse whether the Sorafenib renders FLT3-ITD-positive acute myeloid leukemia (AML) cells more immunogenic . We used Ba/F3-ITD cells as a model cell line to study the effect of Sorafenib on FLT3-ITD-positive AML cells. Ba/F3-ITD cells are murine pro-B cell lines with a stable FLT3-ITD expression. Ba/F3-ITD cells were treated with DMSO or 10nM sorafenib for 24 hours. Cells were harvested and total RNA was isolated
Project description:Ba/F3 mouse cell lines overexpressing or not different types of CALRm (type 1 (del52, del34, del46) and type 2 (del19, ins5)) were starved 6 hours from cytokines and collected. RNA were submitted to microarrays.
Project description:RAS oncogenic mutations are common in human cancers, but RAS proteins have been difficult to target. We sought to identify pharmacological agents to block the RAS oncogenic signaling by a distinct mechanism. Since the biological activity of RAS proteins rely upon lipid modifications and RAS regulates lipid metabolisms in cancer cells, we screened a bioactive lipid library using a RAS specific cell viability assay. We report the discovery of a new class of inhibitors for RAS transformation. Compounds in the class represented by endocannabinoid N-arachidonoyl dopamine (NADA) can induce cell oncosis, independent of its ability to engage cannabinoid receptors. Further analyses show that NADA is more active in inhibiting the NRAS transformation and signaling than that of KRAS4B. Mechanistically, NADA blocks the plasma membrane translocation of NRAS, but not that of KRAS4B. In addition, NADA inhibits the plasma membrane translocation and neoplastic transformation of oncogenic KRAS4A. Interestingly, NADA also redistributes the cytoplasmic NRAS to the Golgi apparatus in a palmitoylation-dependent manner. The results indicate that NADA inhibits NRAS and KRAS4A plasma membrane translocation by targeting a novel molecular process. The new findings would help to develop novel targeted therapies for a broad range of human cancers. To confirm the oncogenic RAS pathway interference of NADA, we performed microarray assay to detail the gene expression pattern change in order to figure out the RAS pathway affection and further indicate the underlying mechnism of the compound function. Overall design: In the microarray assay, two groups were included, which are NRAS-G12D transformed Ba/F3 (Ba/F3-N ) cells treated with vehicle or 1 μM NADA for 12 hours, respectively. Each group contains three replicates. In each sample, 1×107 cells were harvested and for RNA extraction. After quality control, the samples were subjected to the Affymetrix Mouse Genome 430 2.0 Array according to the manufacturer’s instructions.