V3021

Cell-free conditioned medium was generated from each indicated Corynebacterium spp. after inoculation into BHI cultures and growth at 37°C shaking at 200 RPM for 48 h. Conditioned medium was removed after centrifugation of 48-h cultures for 10 m at 7,000 g and then passed through a 0.2 μm filter (Millipore, #SCGP00525) yielding CFCM, which was either used directly for experiments, frozen at -80°C prior to use or further passed through a 3 kDa molecular weight size exclusion filter (Millipore #UFC900324) then frozen at -80°C. For some samples, CFCM was heat treated at 95°C for 15 m. For other samples, CFCM was Proteinase K (Promega #V3021) treated by addition of 100 μg in 1 ml of CFCM prior to incubation at 55°C for 1 h followed by a 95°C heat inactivation for 10 m prior to testing for the effect on AIP-1.

polg2^+/+, polg2^+/ia304 and polg2^ia304/ia304 larvae were collected at 6 dpf and 20 dpf to analyse mtDNA relative quantity by real-time qPCR. While a small section of the tail was cut to perform the genotyping, the fish body was stored in lysis buffer (100 mM Tris HCl pH 8-8.5, 200 mM NaCl, 0.2% SDS, 5 mM EDTA) and proteinase K (10 mg/mL, V3021, Promega), at -20 °C. After the genotyping, individuals were grouped together by genotype, creating pools of 10 fish. Each pool was kept overnight at 55 °C to release the DNA from the cell lysate. Total DNA was extracted by the phenol-chloroform method: 2.5 volumes of 100% EtOH and 1/10 volume of 3 M sodium acetate (NaAc) were used to precipitate DNA. Precipitated DNA was later washed with 500 μL of 70% EtOH, left to dry and re-suspended in 15 μL of nuclease-free water. DNA quantification was performed using the NanoDrop system (NanoDrop 2000, Thermo Scientific). Samples were stored at -20 °C until analysis.