Project description:The discovery of Toll-like receptors (TLRs) represented a significant breakthrough that paved the way for the study of host-pathogen interactions in innate immunity. However, there are still major gaps in understanding TLR function, especially the early dynamics of downstream TLR pathways remains less clear. We characterize a label-free optical biosensor-based assay as a powerful method to detect TLR activation in a native and label-free environment and to delineate the dynamics of TLR pathway activation. To gain a deeper insight into the biological processes underlying the ligand-specific signal traces of different LPS chemotypes in various cell types, RNA-seq analysis of transcriptional changes in HEK Blue hTLR4 reporter cells was performed. After 3 hours of stimulation with LPS E. coli or LPS S. minnesota, HEK Blue hTLR4 reporter cells showed significant changes in RNA expression compared to the unstimulated control.
Project description:The discovery of Toll-like receptors (TLRs) represented a significant breakthrough that paved the way for the study of host-pathogen interactions in innate immunity. However, there are still major gaps in understanding TLR function, especially the early dynamics of downstream TLR pathways remains less clear. We characterize a label-free optical biosensor-based assay as a powerful method to detect TLR activation in a native and label-free environment and to delineate the dynamics of TLR pathway activation. To gain a deeper insight into the biological processes underlying the ligand-specific signal traces of different LPS chemotypes in various cell types, RNA-seq analysis of transcriptional changes in THP-1 macropahges was performed. After 3 hours of stimulation with LPS E. coli or LPS S. minnesota, THP-1 macrophages showed significant changes in RNA expression compared to the unstimulated control.
Project description:In endothelial cells (ECs), stimulation of Toll-like receptor 4 (TLR4) by the endotoxin lipopolysaccharide (LPS) induces the release of diverse pro-inflammatory mediators beneficial in controlling bacterial infections. However, their systemic secretion is a main driver of sepsis and chronic inflammatory diseases. Since distinct and rapid TLR4 signaling induction is difficult to achieve by LPS due to its poorly controllable binding and non-specific affinity to other surface molecules and receptors, we engineered new light-oxygen-voltage-sensing (LOV) domain-based optogenetic endothelial cell lines (opto-TLR4-LOV LEC and opto-TLR4-LOV HUVEC) that allow precise temporal and reversible activation of TLR4 signaling pathways. Using quantitative mass spectrometry, RT-qPCR and Western Blot analysis, we show that pro-inflammatory proteins were not only expressed differently, but also had a different time course when the cells were stimulated with light or LPS. Additional functional assays demonstrated that light induction promoted chemotaxis of THP-1 cells, disruption of the EC-monolayer and transmigration. In contrast, ECs incorporating a truncated version of the TLR4 extracellular domain (opto-TLR4 ΔECD2-LOV LEC) revealed high basal activity with fast exploitation of cell signaling system upon illumination. We conclude that the established optogenetic cell lines are well suited to induce rapid and precise photoactivation of TLR4, allowing receptor-specific studies.
Project description:In order to understand the transcriptional effects of CD44s expression in a cell line that does not express CD44 in its native form we transfected CD44s into HEK cells and measured the transcriptional chances compared to native HEK cells
Project description:Phase separation of biomolecules into condensates is a key mechanism in the spatiotemporal organization of biochemical processes in cells. We systematically engineered light-inducible transcription factor condensates with different material properties and analyzed their influence on transcription activation. When transcription factor condensates were transformed into solid-like gels, we observed a reduced activation of gene expression. We wanted to evaluate the impact that the condensate formation of the transcription factor RelA had on its endogenous promoters using expression data. To this aim, HEK-293T cells were transfected either with empty vector (1-3) or eGFP-RelA (4-6) or eGFP-RelA, Cry2olig-mCh-FUSN-NLS-NbGFP and Cry2olig-mCh-FUSN-NLS, along with an NF-κB-responsive SEAP reporter (7-9). In each of the three groups of transfected cells, 8 h after transfection, 3 samples were kept in the dark (D) and 3 under blue light illumination (BL, 5 μmol m-² s-1) for 24 h prior to RNA extraction. RNA-seq libraries from the 18 samples were sequenced by BGI on a DNBSEQ platform using paired-end chemistry with a read length of 100 base pairs each. Each strand was sequenced across two separate lanes (L03, L04), generating a total of 4 FASTQ files per sample.
Project description:HEK-Blue™ ISG Cells and HEK-Blue™ ISG-KO-STING Cells (Invivogen) were transfected with dacA (Listeria monocytogenes) for 12 and 24 hours. Through this approach, we described the STING-dependent and STING-independent transcriptional response to dacA. Our analysis reveals a STING-dependent enrichment of interferon stimulated genes beginning 12 hours post-transfection, and a STING-independent enrichment of genes associated with protein translation and oxidative phosphorylation.
Project description:In order to understand the transcriptional effects of CD44s expression in a cell line that does not express CD44 in its native form we transfected CD44s into HEK cells and measured the transcriptional chances compared to native HEK cells Three biological replicates from each condition (native HEK cells and CD44s transfected cells) were measured using Affymetrix arrays