Aquasil c18 140 4.6 mm column

Mycelia from 5 mL culture broth were harvested by filtration on filter paper, dried overnight at 100°C and weighed. Filtered medium was passed through a 0.2 μm PVDF membrane (Acrodisc® LC; Pall, Life Sciences) and injected into an AQUASIL C18 140×4.6 mm column (Thermo Scientific) connected to an HPLC device (Agilent 1120 Compact LC) to determine extracellular nucleoside concentrations by monitoring absorbance at 260 nm. The separation of nucleosides was achieved by using an isocratic flow of phosphate buffer, pH 5.5, plus 0.5% of acetonitrile. Quantification was carried out using a calibration curve prepared with pure standards of inosine and guanosine (Sigma-Aldrich). All analyses were performed using three biological replicates.
Metabolism quenching and metabolite extraction were carried out as described [28 (link),29 (link)], using the mycelia obtained from 7 mL of culture broth. Briefly, samples were quenched by the addition of methanol at −40°C, followed by boiling ethanol, and extracted upon the addition of acetonitrile by mechanical homogenization and subsequent chloroform/chloromethane organic extraction steps. The intracellular concentrations of the metabolites in the purine pathway were determined following previously published methodologies [30 (link)].

Extracellular inosine concentration were determined from the A. gossypii culture broth by HPLC. Briefly, mycelia from 5 ml culture broth were harvested by filtration on filter paper, dried overnight at 100 °C and weighed. Filtered medium was passed through a 0.2μm polyvinylidene difluoride membrane (Acrodisc LC; Pall Life Sciences) and injected into an AQUASIL C18 140 × 4.6 mm column (Thermo Fisher Scientific) connected to an HPLC device (Agilent 1120 Compact LC), to determine extracellular nucleoside concentrations by monitoring absorbance at 260 nm. The separation of nucleosides was achieved by using an isocratic flow of phosphate buffer, pH 5.5, plus 0.5% of acetonitrile. Quantification was carried out using a calibration curve prepared with pure standards of inosine (Sigma-Aldrich). All analyses were performed using three biological replicates.