Effect of ∆Np63 deletion on plasticity of post-injury lung basal cells
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ABSTRACT: Basal cells were isolated from an influenza-infected non-tamoxifen treated Krt5CreERT2; RFP; ∆Np63flox/flox mouse and cultured, allowing for inducible ∆Np63 deletion in vitro. These basal cells were plated as monolayers, airway organoids, and alveolar organoids and treated with 1uM 4OHT for +4OHT (∆Np63 KO) or equivalent volume DMSO for -4OHT (solvent only, ∆Np63 WT). Technical triplicates of ∆Np63 WT and ∆Np63 KO were harvested for each growth condition for RNA sequencing.
Project description:Basal cells were isolated from an influenza-infected non-tamoxifen treated Krt5CreERT2; RFP; ∆Np63flox/flox mouse and cultured, allowing for inducible ∆Np63 deletion in vitro. These basal cells were plated as monolayers and treated with 1uM 4OHT for +4OHT (∆Np63 KO) or equivalent volume DMSO for -4OHT (solvent only, ∆Np63 WT) for 48 hours. Technical triplicates of ∆Np63 WT and ∆Np63 KO treated in parallel were harvested for each IP for p63 and each histone modification for ChIP-seq.
Project description:MaSC, luminal progenitor enriched subpopulations and total MECs as well, were isolated from both wild type and ∆Np63 KO heterozygous mouse and the transcriptome profiles were determined and compared. Three populations: P4, P5 and MECs; two genotypes: WT vs ∆Np63 heterozygous.
Project description:MaSC, luminal progenitor enriched subpopulations and total MECs as well, were isolated from both wild type and ∆Np63 KO heterozygous mouse and the transcriptome profiles were determined and compared.
Project description:Distinct lung stem cells give rise to lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC). ΔNp63 guides development of these cells through regulation of terminal differentiation; however, its mechanistic role in lung cancer development has remained elusive. We utilized a ΔNp63-specific conditional knockout mouse model and found that ∆Np63 maintains lung ADC and SCC by keeping lung stem cells in quiescence. ChIP-seq analysis of lung basal cells and alveolar type 2 (AT2) cells lacking ∆Np63 revealed a robust loss of activating histone marks at super enhancers of cell identity genes defining a unifying oncogenic role for ∆Np63 in non-small cell lung cancer.
Project description:The oncoprotein ∆Np63 is an essential transcriptional master- and cell identity regulator in squamous cell carcinoma (SCC) of various origins, encompassing lung, head and neck, oesophagus, cervix and skin. While in non-transformed cells ∆Np63 protein abundance is tightly regulated by the ubiquitin proteasome system (UPS), in tumours E3-ligases ubiquitylating ∆Np63, such as FBXW7, are commonly mutated or lost, resulting in a hyper-stabilisation of the oncogenic driver. Targeting ∆Np63 protein abundance in SCC could present a possible therapeutic avenue. Here, we report that the deubiquitylase USP28 regulates ∆Np63 protein stability and abundance in SCC by counteracting the degradative UPS system. Interference with USP28 activity by pharmacological inhibition specifically affected human SCC cell lines and, finally, we were able to demonstrate in vivo using CRISPR/Cas9 mouse models that Usp28 is required for SCC induction and maintenance. Hence, targeting USP28 is a viable option to tackle ∆Np63 protein abundance in SCC tumours.
Project description:delta-Np63 is highly expressed in squamous cell carcinoma of the head and neck (HNSCC). To evaluate its function in HNSCC we depleted delta-Np63 by siRNAs in the HNSCC cell line UT-SCC-74A. The transcriptome was analysed by cDNA microarray.
Project description:The goal of this study was to perform transcriptomics on wildtype, PTEN single knockout (SKO) and PTEN;Rb1 double knockout (DKO) mouse prostate organoids. We isolated basal cells from PTEN floxed and PTEN;Rb1 floxed mouse prostates and infected with either RFP control or Cre recombinase to establish wildtype, SKO, and DKO mouse prostate organoids.
Project description:Clostridioides difficile (C. difficile) toxins A (TcdA) and B (TcdB) cause antibiotic-associated colitis, increasing morbidity and mortality. Accurate in vitro models are necessary to detect early toxicity kinetics, investigate disease etiology, and develop preclinical models for new therapies. Properties of cancer cell lines and organoids inherently limit these efforts. We developed adult stem cell-derived monolayers of differentiated human colonic epithelium (hCE) with barrier function, investigated the impact of toxin application to apical/basal aspects of monolayers, and evaluated whether a leaky epithelial barrier enhances toxicity. Single-cell RNA-sequencing (scRNAseq) mapped C. difficile-relevant genes to human gut epithelial lineages. Transcriptomics informed timing of stem cell differentiation to achieve in vitro colonocyte maturation like that observed in vivo. Transepithelial electrical resistance (TEER) and fluorescent dextran permeability assays measured cytotoxicity as barrier loss post-toxin exposure. Leaky epithelial barriers were induced with diclofenac. scRNAseq demonstrated broad and variable toxin receptor expression across human gut lineages. Absorptive colonocytes displayed generally enhanced toxin receptor, Rho GTPase, and cell junction expression. 22-day differentiated Caco-2 cells remained immature whereas hCE monolayers were similar to mature colonocytes. hCE monolayers exhibited high barrier function after 1-day differentiation. Basal TcdA/B application to monolayers caused greater toxicity and apoptosis. Diclofenac induced leaky hCE monolayers and enhanced toxicity of apical TcdB exposure. Apical/basal toxicities are uncoupled with more rapid onset and increased magnitude of basal toxicity. Leaky paracellular junctions enhance toxicity of apical TcdB exposure. hCE monolayers represent a physiologically relevant and sensitive culture system to evaluate the impact of microbial toxins on gut epithelium.
Project description:We investigated changes in transcriptome in Egfr KO PSCs co-cultured with pancreatic tumor organoids compared to Egfr WT PSCs co-cultured with the same pancreatic tumor organoids. We also investigated the changes in transcriptome in pancreatic tumor organoids co-cultured with Egfr WT or Egfr KO PSCs.