Project description:In chicken DT40 cells, there are six linker histone H1 variants and 12 of coding genes. We have previously reported of 11 out of 12 H1 knock out DT40 cells (Takami et al., Genes to Cell 1997 [PMID:9491804]) but complete H1 null DT40 cells could not established, so far. We identified one of the H1 variant, H1R was involved in genomic instabilities (Hashimoto et al., DNA repair (2007) [17613284]), so we re-introduced floxed H1R-eGFP and mer-cre-mer into 11 out of 12 H1 knock out DT40 cells. Then we targeted last enedogenous H1, we successfully established conditional H1 KO cells (K11). Next we treated with tamoxifen to loop out floxed H1R-eGFP, and cloning H1 completely null cells (K11-5, and K11-7). We analysis those gene expression pattern in wild-type, K11, and K11-5 cells Experiment Overall Design: Apoptosis is induced in H1 null cells, so we inhibit apoptosis with pan-caspase inhibitor, Z-VAD-FMK and extract RNAs.
Project description:In vitro models are widely used in fundamental research and high-throughput drug discovery. Particularly the sensory neurons dissociated from dorsal root ganglion (DRG) are cultured in monolayer to study the pain associated biology. The problem of monolayer culture is that the naïve multicellular architecture is deprived. The impact of multicellular assembly on biology remains elusive. Also, biofabrication of multicellular system usually depends on a complex scaffold design to support the aggregate which is difficult to scale up. The scaffold-cell interaction may confound the in vitro testing result. Here, we present a scaffold-free assembly using hydrodynamic forces. Cells are assembled in normal culture medium into a precise geometry within two minutes. During culture, the DRG cells self-organize into a 2.5 ~ 3-dimensional architecture. Cells binds to each other to maintain their shape and mechanobiology. We demonstrate interneuron crosstalk in the multicellular system via the synchronization of calcium signals and the cluster-wise expression of neuronal activation markers. Thus, multicellular architecture determines the cell shape and functional crosstalk. This evidence pinpoints the multicellular principle of in vitro models. The scaffold-free assembling is a simple tool to improve the robustness of in vitro model in predicting in vivo biology, which may benefit the innovation in pain medicine.
Project description:The endoplasmic reticulum (ER) is an organelle that performs a variety of essential cellular functions via interactions with other organelles. Despite its important role, chemical tools for profiling the composition and dynamics of ER proteins remain very limited because of the labile nature of these proteins. Here, we developed ER-localizable reactive molecules (called ERMs) as tools for ER-focused chemical proteomics. ERMs can spontaneously localize in the ER of living cells and selectively label ER-associated proteins with a combined affinity and imaging tag, enabling tag-mediated ER protein enrichment and identification with liquid chromatography tandem-mass spectrometry (LC-MS/MS). The ERM probes could be used simultaneously with the nucleus- and mitochondria-localizable reactive molecules previously developed by our group, which enabled orthogonal organellar chemoproteomics in a single biological sample. Moreover, quantitative analysis of the dynamic changes in ER-associated proteins in response to tunicamycin-induced ER stress was performed by combining ER-specific labeling with SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative MS technology. Our results demonstrated that ERM-based chemical proteomics provides a powerful tool for labeling and profiling ER-related proteins in living cells.
Project description:We devised a novel methodology for sorting stroma-free tumor cells according to their relative distance from BVs by injecting a vascular perfusion marker (Hoechst 33342). Briefly, post 4 weeks of sub cutaneous implantation of U87MG-GFP cell line into dorsal flank of NOD-SCID mice, the mice were injected i.v. with Hoechst dye. Mice sacrificed, tumors retrieved, single cell dissociated and FACS sorted to isolate cells located at progressive distance from blood vessels based on perfusion dye intake. Three fractions namely - Hh (close to BVs), Hm (intermediate) and Hl (farther from BVs) were FACS sorted. RNA was extracted from the sorted cells and RNA-Sequencing performed.
Project description:Members of the chromodomain-helicase-DNA binding (CHD) protein family are chromatin remodelers implicated in human pathologies, with CHD6 being one of its least studied members. We discovered a de novo CHD6 missense mutation in a patient clinically presenting the rare Hallermann-Streiff syndrome (HSS). We used genome editing to generate isogenic iPSC lines and model HSS in relevant cell types. By combining genomics with functional in vivo and in vitro assays, we show that CHD6 binds a cohort of autophagy and stress response genes across cell types. The HSS mutation affects CHD6 protein folding and impairs its ability to recruit co-remodelers in response to DNA damage or autophagy stimulation. This leads to accumulation of DNA damage burden and senescence-like phenotypes. We therefore uncovered a molecular mechanism explaining HSS onset via chromatin control of autophagic flux and genotoxic stress surveillance.
Project description:Targeted therapies against cancer stem cells which are enriched in side populations (SP) involves interruption of Wnt-signalling. Furthermore, EpCAM is a SP marker and modulator of Wnt-signalling. Therefore, the effects of an anti-EpCAM treatment on SP-cells and WNT/M-NM-2-catenin signalling was studied. SP of the murine lung adenocarcinoma cell line A2C12 was obtained by fluorescence activated cell sorting and whole genome scans helped to define their molecular phenotype after anti-EpCAM antibody treatment. Anti-EpCAM treated and untreated A2C12 cells were subjected to Hoechst 33342 dye exclusion assay and sorted to SP and non-SP fractions by FACS. Three biological replicates.
