Study design
Cells and cell cultures
Mouse neonatal fibroblast
Plasmids
Viral particle production and infection
HSV plaque assay
Protein purification
Nuclear cytoplasmic fractionation
Biotinylated nucleic acid pull-down using recombinant protein
Biotinylated nucleic acid pull-down using cell extracts
In vitro biotinylated nucleic acid pull-down
Pull-down using biotinylated Ap4A and recombinant proteins
S9.6 immunoprecipitation
RNA extraction and RT-qPCR
Whole-cell extract preparation and immunoblot
Morpholino knock-down experiments
Molecular modeling
cGAMP ELISA
Radiolabeling
Cell treatment and transfection
Immunofluorescence and microscopy analysis
Statistical analysis
Compounds (PubChem CID)
Oligonucleotide sequences
Morpholino sequences
Oligo sequences for annealing
shRNA sequences
Cellular nucleotide extraction and Ap4A determination were performed using a sensitive luminescence-based assay. Briefly, nucleotides were extracted from cells by adding 5 ml of ice-cold trichloroacetic acid and subsequent neutralization with an equal volume of 0.6 M tri-octylamine in 1,1,2-trichlorotrifluoroethane. Following centrifugation at 500g for 5 min, the top aqueous layer was removed, and 10 U of thermo-sensitive alkaline phosphatase (Thermo Fisher Scientific) was added to degrade adenosine triphosphate (ATP). Remaining nucleotides were concentrated using DEAE-Sephacel beads. Following mixing and centrifugation at 12,000g for 1 min, beads were washed with water. Nucleotides bound to DEAE-Sephacel were eluted twice with 1 M triethylammonium bicarbonate (pH 8.5), and eluates were vacuum-dried. Nucleotides from the two eluates were combined and dissolved in 120 μl of Ap4A assay buffer [25 mM Hepes-NaOH (pH 7.8) and 5 mM magnesium acetate]. To remove any remaining ATP, a further 10 U of alkaline phosphatase was added, incubated at 37°C, and subsequently incubated at 95°C to denature alkaline phosphatase. Ap4A levels in 10 μl of sample were determined by adding 50 μl of Bactiter GLO (Promega). Background ATP levels were first measured on a Berthold Lumat 9507, and once stabilized, recombinant Ap4A hydrolase was added to cleave Ap4A (ATP + AMP), and levels of ATP were measured by the increase in luminescence. Levels of Ap4A in samples were determined by comparison to standards and expressed as pmol per 106 cells.