D. melanogaster cyclic dinucleotide injection and signalling analysis

Fly stocks were raised on standard cornmeal agar medium at 25 °C. All fly lines used in this study were Wolbachia free. w¹¹¹⁸, dSTING^Control and dSTING^Rxn stocks have been described previously^23,26. Relish^E20 flies isogenized to the DrosDel w¹¹¹⁸ isogenic background were a kind gift from L. Teixeira (Instituto Gulbenkian de Ciência)⁴⁶. Cyclic dinucleotides including 3′2′-cGAMP (Biolog), 2′3′-cGAMP (Invivogen) and 3′3′-c-di-GMP (Invivogen) were dissolved in 10 mM Tris-HCl pH 7.5 and diluted to the indicated concentrations. Adult flies (3–5-day old) were injected with 69 nl of cyclic dinucleotide solution or 10 mM Tris-HCl pH 7.5 (negative control) by intrathoracic injection using a Nanoject II apparatus (Drummond Scientific). Flies were collected 24 h later in pools of 6 individuals (3 males and 3 females) or 10 individuals (5 males and 5 females) and homogenized for RNA extraction and quantitative PCR with reverse transcription (RT–qPCR) analysis, as described²⁶. The sample size for all Drosophila experiments was determined using previously published protocols²⁶. Flies were randomly selected for each experimental group and blinding was not performed.