Focal amplification of HOXD-harboring chromosome region is implicated in multiple-walled carbon nanotubes-induced carcinogenicity
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ABSTRACT: Amplificaition of HOXD9 and HOXD13 genes was found in MWCNTs induced carcinogencity. By overexpression or silence of of HOXD9 and HOXD13 gene may alter tumorigenicity. To investigate the realted signaling pathways invovled in HOXD regulated tumorigencity, we analyzed the gene expression profile of the HOXD9 or HOXD13 overexpression HEK293 cells, which compared to vehicle expressing cells HEK293 cells was transfected with HA-tag-HOXD9 and V5-tag HOXD13 vector, and further selected by G418. Three independnent clones and mix clones of each genes were selected, and compared to corresponding tag control cell. (HA-HOXD9-mix, a, b, c v.s HA-MOCK-1, 2, 3) (V5-HOXD13-mix, a, b, c v.s V5-MOCK-1, 2, 3)
Project description:Compound heterozygous or homozygous GBA1 mutations lead to Gaucher disease. Furthermore, GBA1 mutations are the most frequent risk factor for Parkinson’s disease. To get a better understanding of the pathological mechanisms, we generated inducible V5-Flag-Tag, V5-Flag-tagged WT, E326K and L444P mutant Flp-In™T-REx™-HEK293 and performed interatomic analysis.
Project description:This project aims to identify novel regulators of mtDNA turnover, for what we used TurboID. For specific detection of the mitochondria-endosome proteome, we fused the N-terminal part of Turbo ID to RAB5C (TurboID aa1-aa72; RAB5C-SplitTurboNt-V5) and, the C-terminal part to SAMM50 (TurboID aa73–aa246; SAMM50-SplitTurboCt-HA). Proximity biotinylation was performed in transduced HEK293 cells expressing Split TurboID constructs. Cells expressing only SAMM50-SplitC-HA were used as a negative control. For protein isolation, HEK293 cells were incubated for 4 h with 0,25 mM biotin. For inducing mtDNA damage, cells were previously transfected with Twinkle K319E-Cherry 24 h before the biotin incubation.
Project description:To validate role of REST (RE-1 silencing transcription factor) in glioblastoma (GBM) growth, we used CRISPR/Cas9 gene editing to generate REST-null single-cell clonal lines from GBM cell line (T98G) and non-neural HEK293 cells. We then performed gene expression profiling using data obtained from Tag-Seq of 8 cell lines: T98G CRISRP Control and 4 T98G REST-KO clones (C10, F7, G2, D4); HEK293 CRISPR Control and 2 HEK293 REST-KO clones (D10, E6).
Project description:To demonstrate RIPSeeker program that is developed for RIP-seq analyses, we generated RIP-seq data corresponding to the protein CCNT1 in HEK293 cell line using standard RIP-seq protocols described in Zhao et al., (2010). We performed two in-house RIP-seq experiments both for CCNT1 in human HEK293 cells. Briefly, we generated tagged CCNT1 using a triple tag system that supports lentiviral stable expression and mammalian affinity purification (MAPLE) Mak et al (2010). The HEK293 cells stably expressing tagged CCNT1 was purified by M2 agarose beads, followed by RNA extraction by Trizol. The library synthesis was carried out according to the RIP-seq protocol described in Zhao et al., (2010) except that one of the two experiments was done with non-strand-specific sequencing.
Project description:Amplificaition of HOXD9 and HOXD13 genes was found in MWCNTs induced carcinogencity. By overexpression or silence of of HOXD9 and HOXD13 gene may alter tumorigenicity. To investigate the realted signaling pathways invovled in HOXD regulated tumorigencity, we analyzed the gene expression profile of the HOXD9 or HOXD13 overexpression HEK293 cells, which compared to vehicle expressing cells
Project description:Cut & Run analysis was performed in an neuroblastoma cell line to analyze DNA bindings of ASCL1-tag-HA in GI-MEN ASCL1-tag-HA cells and GI-MEN ASCL1-tag-HA+4TFs cells; analyze DNA bindings of MYCN, PHOX2B and H3K27ac in, GI-MEN 4TFs cells, and GI-MEN ASCL1-tag-HA+4TFs cells.
Project description:Meiotic chromosomes are highly compacted yet remain transcriptionally active. To understand how chromosome folding accommodates transcription, we investigated the assembly of the axial element, the proteinaceous structure that compacts meiotic chromosomes and promotes recombination and fertility. We found that the axial element proteins of budding yeast are flexibly anchored to chromatin by the ring-like cohesin complex and biased towards small chromosomes by a separate modulating mechanism that requires the conserved axial-element component Hop1. The ubiquitous presence of cohesin at sites of convergent transcription provides well-dispersed points for axis attachment and thus compaction. Axis protein enrichment at these sites directly correlates with the propensity for recombination initiation. Importantly, axis anchoring by cohesin is adjustable and readily displaced in the direction of transcription by the transcriptional machinery. We propose that such robust but flexible tethering allows the axial element to promote recombination while easily adapting to changes in chromosome activity. 7 genome wide meiotic ChIP-seq sets: V5-Red1 DNA interaction (V5-Red1-ChIP), V5-Red1 DNA interaction in the absence of axis protein Hop1 (V5-Red1-ChIP, hop1delta), V5-Red1 DNA interaction in the absence of another two axis proteins Hop1 and Rec8 (V5-Red1-ChIP, hop1delta rec8delta), Rec8-HA DNA interaction (Rec8-HA-ChIP), Rec8-HA DNA interactionin the absence of Red1 (Rec8-HA-ChIP, red1delta), and 2 untagged control (V5-untagged-ChIP, HA-untagged-ChIP) (corresponding to the main Figure5)
Project description:T-HF cells were grown either in the presence or absence of DOX. Upon DOX exposure, cells expressed either HA-ICP22 or HA-ICP22 and V5-ICP27. T-HF HA-ICP22 cells were treated with salt stress for 2 hours before proceeding with Omni-ATAC-seq. OMNI-ATAC-seq was conducted for the KOS1.1 strain and compared to a full US1 deletion mutant.
Project description:A stable HEK293 FlpIn T-Rex cells expressing TDP-43 with an N-terminal eGFP-tag was generated that allowed inducible physiological expression of the protein (Ling et al. 2010). Duplicate iCLIP experiments were performed using an antibody targeting eGFP (Abcam ab290). Crosslinked RNA-protein complexes were isolated by immuno-precipitation and cDNAs were generated to allow preparation of Illumina compatible DNA libraries as described in Huppertz et al. (2014).