Shown is a representative blot from three biological replicates.Wild type and STING strains were treated with increasing concentrations of cGAMP, and survival was quantified after hr.In contrast to wild type cells, cGAMP does not induce cell death in STING cells.Data represent mean SD for four biological replicates.Wild type and STING cells were transfected with STING mTFP, and treated with puromycin to generate stable clonal strains.Stable expression of STING mTFP in STING cells partially <a href="https://www.targetmol.com/compound/AMG%20837%20calcium%20hydrate">buy AMG 837</a> rescued the phenotype of cGAMP induced cell death.Data represent mean SD for three biological replicates.Differential expression analysis was performed on wild type and STING cells treated with M cGAMP or a vehicle control for hr.A heatmap comparing the log fold change of genes identified as differentially expressed in wild type cells after cGAMP treatment, to their log fold change in STING cells after cGAMP treatment.RNA seq libraries were prepared from two biological replicates.Presence of STING in the transcriptomes of diverse choanoflagellate species.Wild type and STING cells have similar survival responses to LPS, suggesting that STING is not required for mediating a response to LPS.Data represent mean SD for four biological replicates.These results suggest that treatment with cGAMP induces autophagic signaling in a STING dependent manner; however, making this conclusion requires evidence of autophagy induction through inhibitor studies.In cells pretreated with chloroquine, STING levels did not markedly increase after exposure to cGAMP, raising the possibility that the autophagic pathway is important for regulating STING protein levels.Finally, we asked whether STING mediated autophagic pathway induction affects survival after exposure to cGAMP. Treatment with lysosomotropic agents that inhibit autophagy rescue cGAMP induced cell death in wild type cells.Cells were then <a href="https://www.ncbi.nlm.nih.gov/pubmed/22217876">AMG 837</a> exposed to either M cGAMP or a vehicle control for hr before quantifying survival.Data represent mean SD for three biological replicates.Two pathways downstream of STING activation that are conserved in invertebrates, and as such are proposed ancestral functions of STING, are autophagy and NF kB signaling.Choanoflagellates forage on diverse environmental bacteria for sustenance, yet how they recognize and respond to pathogens is a mystery.Identifying additional choanoflagellate pathogens, particularly viral pathogens, will also be key to delineating immune response pathways.Finally, as choanoflagellates are at least as genetically diverse as animals, expanding studies of immune responses to diverse choanoflagellate species will be essential for reconstructing the evolution of immune pathways in animals.Cells were grown either at room temperature, or at C in a wine cooler. After isolating individual colonies, partial S sequencing using S universal primers was used to determine the identity of the bacterial isolates.For each bacterium, a single colony was inoculated into LB and grown shaking overnight at either C. Bacterial cells were pelleted by centrifugation for minutes at xg, and resuspended in artificial seawater to an OD.For each bacterial strain, CFU plating was used to estimate the number of bacterial cells mL under these growth conditions.At least three biological replicates are represented in each graph.For each bacterial strain, CFU plating was used to estimate the number of bacterial cells mL under these growth conditions.mm sterilizing filters.To determine if the bioactivity of the conditioned media is due to a protein, conditioned media was incubated with gmL proteinase K at C for hr, after which the enzyme was deactivated by heating the conditioned media to C for min.
