Anti-LRR1 monoclonal antibody Made to order

R-Spondins are secreted glycoproteins (RSPO1-RSPO4) that have proliferative effects on adult stem cells by potentiating Wnt signaling. RSPO actions are mediated by the leucine-rich repeat (LRR)-containing seven-transmembrane receptors LGR4-LGR6 and the transmembrane E3 ubiquitin ligases ZNRF3 and RNF43. Here, we present a methodology for the bacterial expression and purification of the signaling competent, cysteine-rich Fu1-Fu2 domains of the four human RSPOs, a fragment of the human LGR4 extracellular domain (ECD) containing LRR1- 14, and the human ZNRF3 ECD. In a cell-based signaling assay, the nonglycosylated RSPOs enhanced low-dose Wnt3a signaling with potencies comparable to those of mammalian cell-produced RSPOs and RSPO2 and -3 were more potent than RSPO1 and -4. LGR4 LRR1-14 and ZNRF3 ECD inhibited RSPO2-enhanced Wnt3a signaling. The RSPOs bound LGR4 LRR1-14 with nanomolar affinities that decreased in the following order in a time-resolved fluorescence resonance energy transfer (TR-FRET) assay: RSPO4 > RSPO2 > RSPO3 > RSPO1. RSPO receptor interactions were further characterized with a native gel electrophoretic mobility shift assay, which corroborated the RSPO-LGR4 TR-FRET results and indicated that RSPOs weakly bound ZNRF3 with affinities that decreased in the following order: RSPO2 > RSPO3 > RSPO1. RSPO4:ZNRF3 complexes were not detected. Lastly, ternary RSPO:LGR4:ZNRF3 complexes were detected for RSPO2 and -3. Our results indicate that RSPO and LGR4 N-glycans are dispensable for function, demonstrate RSPO-mediated ternary complex formation, and suggest that the stronger signaling potencies of RSPO2 and -3 result from their strong binding of both receptors. Our unique protein production methodology may provide a cost-effective source of recombinant RSPOs for regenerative medicine applications.

The aim of the present study was to investigate the mechanisms of long non-coding RNAs (lncRNAs) in a gastric cancer cell line treated with celecoxib. The human gastric carcinoma cell line NCI-N87 was treated with 15 mu M celecoxib for 72 h (celecoxib group) and an equal volume of dimethylsulfoxide (control group), respectively. Libraries were constructed by NEBNext Ultra RNA Library Prep kit for Illumina. Paired-end RNA sequencing reads were aligned to a human hg19 reference genome using TopHat2. Differentially expressed genes (DEGs) and lncRNAs were identified using Cuffdiff. Enrichment analysis was performed using GO-function package and KEGG profile in Bioconductor. A protein-protein interaction network was constructed using STRING database and module analysis was performed using ClusterONE plugin of Cytoscape. ATP5G1, ATP5G3, COX8A, CYC1, NDUFS3, UQCRC1, UQCRC2 and UQCRFS1 were enriched in the oxidative phosphorylation pathway. CXCL1, CXCL3, CXCL5 and CXCL8 were enriched in the chemokine signaling and cytokine-cytokine receptor interaction pathways. ITGA3, ITGA6, ITGB4, ITGB5, ITGB6 and ITGB8 were enriched in the integrin-mediated signaling pathway. DEGs co-expressed with lnc-SCD-1:13, lnc-LRR1-1:2, lnc-PTMS-1:3, lnc-S100P-3:1, lnc-AP000974.1-1:1 and lnc-RAB3IL1-2:1 were enriched in the pathways associated with cancer, such as the basal cell carcinoma pathway in cancer. In conclusion, these DEGs and differentially expressed lncRNAs may be important in the celecoxib treatment of gastric cancer.