Fig. 1. Caspofungin treatment induces 5,8-diHODE and 8-HODE production by PpoA.

a Representative DAPI channel images of hyphae treated with 1% DMSO, 1 µg/mL caspofungin, 1 µg/mL 8-HODE, or 1 µg/mL 5,8-diHODE grown for 20 h is liquid GMM at 37 °C. Twenty-hour old hyphae grown in a cover glass bottom 96-well plate were stained with CFW and rinsed twice with sterile PBS before fluorescent imaging. Scale bars represent 50 microns. Micrographs are representative of three independent experiments. Northern blot analysis of ppoA expression in (b) WT Af293 and ∆ppoA strains or (c) WT CEA10 under treatment with 0.025% DMSO vehicle or 1 µg/mL caspofungin. Total RNA was extracted from replicate cultures grown in liquid GMM at 250 RPM for 18.5 h before the addition of DMSO or CSPF for 90 min. Loading of 25 µg RNA per sample is shown by ethidium bromide staining of rRNA. 5,8-diHODE and 8-HODE per milligram of dry biomass extracted from (d) fungal tissue or (e) culture supernatant. WT A. fumigatus Af293 was grown at 37 °C and 250 RPM for 24 h in GMM plus 48 h more after the addition of 0.02% DMSO or 2 µg/mL CSPF. Oxylipins were extracted using mixed organic solvent and quantified on UHPLC-MS/MS by comparison to standard curves of purified oxylipin. Data points represent independent culture flasks (n = 3 or 4) and error bars denote SEM. P values shown were calculated using Welch’s two-sided t-test.