Optimization of cDNA microarrays procedures using criteria that do not rely on external standards-5

<p><b>Copyright information:</b></p><p>Taken from "Optimization of cDNA microarrays procedures using criteria that do not rely on external standards"</p><p>http://www.biomedcentral.com/1471-2164/8/377</p><p>BMC Genomics 2007;8():377-377.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2147032.</p><p></p

Optimization of cDNA microarrays procedures using criteria that do not rely on external standards-4

<p><b>Copyright information:</b></p><p>Taken from "Optimization of cDNA microarrays procedures using criteria that do not rely on external standards"</p><p>http://www.biomedcentral.com/1471-2164/8/377</p><p>BMC Genomics 2007;8():377-377.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2147032.</p><p></p> lines NRK52E and AR42J) compared to self-self hybridization (rat cell line AR42J). The samples were hybridized to rat 15 k cDNA duplicates under six different blocking conditions including no blocker, 1000 ng poly(dA), and 25 to 1000 ng LNA dT blocker. Dye-swap and self self were performed for all blocking conditions (total of 24 hybridizations). Green-labelled samples are placed at the tail and red labelled samples at the head of the arrows

Optimization of cDNA microarrays procedures using criteria that do not rely on external standards-1

<p><b>Copyright information:</b></p><p>Taken from "Optimization of cDNA microarrays procedures using criteria that do not rely on external standards"</p><p>http://www.biomedcentral.com/1471-2164/8/377</p><p>BMC Genomics 2007;8():377-377.</p><p>Published online 18 Oct 2007</p><p>PMCID:PMC2147032.</p><p></p

Image_1_Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids.tiff

BackgroundThe epithelium in the colonic mucosa is implicated in the pathophysiology of various diseases, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial organoids from the colon (colonoids) can be used for disease modeling and personalized drug screening. Colonoids are usually cultured at 18-21% oxygen without accounting for the physiological hypoxia in the colonic epithelium (3% to MethodsGrowth from single cells to differentiated colonoids was monitored by brightfield images and evaluated with a linear mixed model. Cell composition was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing (scRNA-seq). Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data.ResultsColonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. No differences in expression of cell markers for cells with proliferation potential (KI67 positive), goblet cells (MUC2 positive), absorptive cells (MUC2 negative, CK20 positive) and enteroendocrine cells (CGA positive) were found between colonoids cultured in 2% and 20% oxygen. However, the scRNA-seq analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CXCL2, CXCL5, CXCL10, CXCL12, CX3CL1 and CCL25, and NGAL upon TNF + poly(I:C) treatment, but there appeared to be a tendency towards lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks.ConclusionsOur results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.</p

Image_2_Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids.tiff

BackgroundThe epithelium in the colonic mucosa is implicated in the pathophysiology of various diseases, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial organoids from the colon (colonoids) can be used for disease modeling and personalized drug screening. Colonoids are usually cultured at 18-21% oxygen without accounting for the physiological hypoxia in the colonic epithelium (3% to MethodsGrowth from single cells to differentiated colonoids was monitored by brightfield images and evaluated with a linear mixed model. Cell composition was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing (scRNA-seq). Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data.ResultsColonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. No differences in expression of cell markers for cells with proliferation potential (KI67 positive), goblet cells (MUC2 positive), absorptive cells (MUC2 negative, CK20 positive) and enteroendocrine cells (CGA positive) were found between colonoids cultured in 2% and 20% oxygen. However, the scRNA-seq analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CXCL2, CXCL5, CXCL10, CXCL12, CX3CL1 and CCL25, and NGAL upon TNF + poly(I:C) treatment, but there appeared to be a tendency towards lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks.ConclusionsOur results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.</p

Table_1_Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids.xlsx

BackgroundThe epithelium in the colonic mucosa is implicated in the pathophysiology of various diseases, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial organoids from the colon (colonoids) can be used for disease modeling and personalized drug screening. Colonoids are usually cultured at 18-21% oxygen without accounting for the physiological hypoxia in the colonic epithelium (3% to MethodsGrowth from single cells to differentiated colonoids was monitored by brightfield images and evaluated with a linear mixed model. Cell composition was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing (scRNA-seq). Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data.ResultsColonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. No differences in expression of cell markers for cells with proliferation potential (KI67 positive), goblet cells (MUC2 positive), absorptive cells (MUC2 negative, CK20 positive) and enteroendocrine cells (CGA positive) were found between colonoids cultured in 2% and 20% oxygen. However, the scRNA-seq analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CXCL2, CXCL5, CXCL10, CXCL12, CX3CL1 and CCL25, and NGAL upon TNF + poly(I:C) treatment, but there appeared to be a tendency towards lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks.ConclusionsOur results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.</p

Expression of CCL20 and Its Corresponding Receptor CCR6 Is Enhanced in Active Inflammatory Bowel Disease, and TLR3 Mediates CCL20 Expression in Colonic Epithelial Cells

<div><p>Background</p><p>The chemokine CCL20 and its receptor CCR6 are putative drug targets in inflammatory bowel disease, and CCL20 is a novel IBD predilection gene. Previous findings on the CCL20 response in these diseases are divergent. This study was undertaken to examine CCL20 and CCR6 during active and inactive disease, and mechanisms for CCL20 regulation by the innate immune system. As TLR3 has recently emerged as a possible mediator of CCL20 production, we hypothesised that this TLR plays an important role in enterocytic CCL20 production.</p><p>Methods</p><p>A large microarray study on colonic pinch biopsies from active and inactive ulcerative colitis and Crohn’s disease provided background information. CCL20 and CCR6 were localized and their expression levels assessed in biopsies using <i>in situ</i> hybridization and immunohistochemistry. Regulation of CCL20 was studied in the HT29 cell line using a panel of pattern recognition receptor ligands followed by a TLR3 siRNA assay.</p><p>Results</p><p><i>CCL20</i> and <i>CCR6</i> mRNA abundances were increased during active inflammation (<i>CCL20</i> 5.4-fold in ulcerative colitis and 4.2-fold in Crohn’s disease; <i>CCR6</i> 1.8 and 2.0, respectively). <i>CCL20</i> and <i>CCR6</i> mRNA positive immune cells in lamina propria were more numerous, and CCL20 immunoreactivity increased massively in the epithelial cells during active inflammation for both diseases. TLR3 stimulation potently induced upregulation and release of CCL20 from HT29 cells, and <i>TLR3</i> silencing reduced CCL20 mRNA and protein levels.</p><p>Conclusions</p><p>The CCL20-CCR6 axis is involved during active inflammation in both ulcerative colitis and Crohn’s disease. The epithelial cells seem particularly involved in the CCL20 response, and results from this study strongly suggest that the innate immune system is important for activation of the epithelium, especially through TLR3.</p></div

Localization of <i>CCR6</i> mRNA in colonic biopsies.

<p>In situ hybridization of <i>CCR6</i> mRNA in colonic inflammatory bowel disease tissue. Sections are taken from biopsies active (UCa) or inactive (UCi) ulcerative colitis, and active (CDa) or inactive (CDi) Crohn’s disease. High and low expression of <i>CCR6</i> in inactive disease is shown. Scale bars as indicated.</p

<i>CCL20</i> and <i>CCR6</i> gene expression in colonic biopsies.

<p><b>A and B:</b> Microarray gene expression results of <i>CCL20</i> and <i>CCR6</i> in colonic biopsies from healthy controls (N), active (UCa) or inactive (UCi) ulcerative colitis, and active (CDa) or inactive (CDi) Crohn’s disease. Individual values (Log₂) and mean are plotted. <b>C and D:</b> qRT-PCR gene expression results of <i>CCL20</i> and <i>CCR6</i> in colonic biopsies from N, Ulcerative Colitis (UC) and Crohn’s disease (CD), n = 5 in each group. Individual values (foldchange 2^-ΔΔCt) and mean are plotted. *p<0.05 versus N, **p<0.01 versus N, ***p<0.001 versus N, ###p<0.001 versus inactive disease.</p

SIK1 inhibits migration in AGS-G_R cells via suppression of MMP-9.

<p><b>A–B:</b> AGS-G_R cells (<b>A</b>) and MKN45 cells (<b>B</b>) were treated with gastrin, and phospho-LKB1 (Ser-428) protein levels determined by Western blot. The phospho-LKB1 bands from a representative experiment are shown. <b>C–D:</b> AGS-G_R cells (<b>C</b>) and MKN45 (<b>D</b>) were treated with gastrin, and phospho-SIK1 (Thr-182) protein levels determined by Western blot. The phospho-SIK1 bands from a representative experiment are shown. <b>E:</b> AGS-G_R cells transfected with siSIK1 or siCtr and real-time cell migration monitored (0–24 h). Results show one representative of three independent experiments (mean ±SD of three technical replicates). <b>F:</b> MMP-9 mRNA expression in cells transfected with pSIK1 and treated with gastrin. Results show one representative of three independent experiments, (mean ± SD).</p