Eflornithine

Effects of eflornithine (Sigma-Aldrich) on fungal growth were analyzed by agar diffusion assays, which were carried out as described previously by Beckmann et al. (2013 (link)), with slight modifications. As the mutant strain analyzed in this study was not arginine auxotrophic, arginine supplementation was omitted.

1 mL cultures of EP1/GFP reporter cells were plated at 100,000 cells/ml and treated with 33 μM of eflornithine (Sigma-Aldrich 1234249), phenothiazine (Sigma-Aldrich P14831), spironolactone (Sigma-Aldrich S3378), or vehicle control and counted daily by hemocytometer. Each day following counting, cells were diluted back to 100,000 cells/ml. For growth curve analysis by flow cytometry, 200 μl cultures of parasites were plated at 100,000 cells/ml and treated with 33 μM of phenothiazine (Sigma-Aldrich P14831), triprolidine hydrochloride (Sigma-Aldrich T6764), flunarizine hydrochloride (Sigma-Aldrich F8257), aprepitant (Sigma-Aldrich SML2215), bufexamac (Sigma-Aldrich B0760), clemastine fumarate salt (Sigma-Aldrich SML0445) or vehicle control. Parasites were stained daily with Sytox Orange and a fixed volume of cells was analyzed by flow cytometry. The number of live cells was calculated as the number of Sytox Orange negative cells falling within the live gate. Each day following flow analysis, cells were diluted back to 100,000 cells/ml according to the number of live cells counted.